Gene related to migraine in man

ABSTRACT

Genes for familial hemeplegic migraine (FHM), episodic ataxia type-2 (EA-2), common forms of migraine, and other episodic neurological disorders, such as epilepsy, have been mapped to chromosome 19p13. A brain-specific P/Q type calcium channel subunit gene, covering 300 kb with 47 exons is provided. The exons and their surroundings reveal polymorphic variations and deleterious mutations that are linked to various types of cation channel dysfunctions causing episodic neurological disorders in man or animals.

Migraine is a frequent paroxysmal neuro-vascular disorder, characterized by recurrent attacks of disabling headache, vomiting, photo/phonophobia, malaise, and other general symptoms (migraine without aura). Up to 20% of patients may, in addition, experience transient neurological (aura) symptoms during attacks (migraine with aura) (HCC, 1988). Up to 24% of females and 12% of males in the general population are affected, however with variable attack frequency, duration and severity (Russell et al., 1995). Knowledge about the mechanisms of the final common pathway of migraine attacks has increased substantially the last five years, resulting in improved, though still only symptomatic (and sub-optimal) acute treatment for the attack. There is, however, still very little knowledge about the etiology of migraine attacks, i.e. why and how attacks begin and recur. Accordingly, prophylactic treatment for migraine is non-specific and has only limited efficacy.

Family, twin and population-based studies suggest that genetic factors are involved In migraine, most likely as part of a multifactorial mechanism (reviewed by Haan et al., 1996). The complex genetics has hampered identification of candidate genes for migraine. Familial Hemiplegic Migraine (FHM) is a rare, autosomal dominant, subtype of migraine with aura, associated with ictal hemiparesis and, in some families cerebellar atrophy (HCC, 1988). Otherwise, the symptoms of the headache and aura phase of FHM and “normal” migraine attacks are very similar and both types of attacks may alternate within subject and co-occur within families. FHM is thus part of the migraine spectrum and can be used as a model to study the complex genetics of the more common-forms of migraine (Haan et al., 1996). A gene for FHM has been assigned to chromosome 19p13 in about half of the families tested (Joutel et al., 1993; Ophoff et al., 1994; Joutel et al., 1995). Remarkably, cerebellar atrophy was found only in families with FHM linked to chromosome 19p13, but not in unlinked families. Recently, we showed the 19p13 FHM locus to be also involved in “normal” migraine (May et al., 1995).

Episodic ataxia type 2 (EA-2) is another, autosomal dominant, paroxysmal neurological disorder, characterized by acetazolamide-responsive attacks of cerebellar ataxia and migraine-like symptoms, and interictal nystagmus and cerebellar atrophy. Recently, a gene for EA-2 was assigned to chromosome 19p13, within the same interval as for FHM (Kramer et al., 1995). This finding, as well as the clinical similarities, raise the possibility of EA-2 and FHM being allelic disorders.

Since other hereditary episodic neurological disorders responding to acetazolamide (such as hypokalaemic and hyperkalaemic periodic paralysis), as well as EA type-1 A (which, in contrast to EA-2, is associated with continuous myokymia and non-responsive to acetazolamide) have all been associated with mutations in genes encoding for ion channels (Ptacek et al., 1991; Ptacek et al., 1994; Brown et al., 1994), we specifically looked for similar genes within the FHM and EA-2 candidate region.

In view of the above, the FHM/EA-2 locus can, since FHM is part of the migraine spectrum, thus be used to study the genetic factors and biological mechanisms that are related to various episodic neurological disorders such as FHM, EA-2, common migraine and others such as epilepsy.

Calcium channels are multisubunit complexes composed of at least an α1, an α2δ, and a β subunit. The central α1 subunit is functionally the most important component, acting as a voltage sensor and forming the ion-conducting pore. The other subunits have auxiliary regulatory roles. The membrane topology of the α1 subunit consist of four hydrophobic motifs (I to IV), each containing six transmembrane α-helices (S1-S6) and one hairpin (P) between S5-S6 that spans only the outer part of the transmembrane region.

The present invention provides an isolated and/or recombinant nucleic acid, or fragments thereof, encoding a Ca²+-channel α1 subunit related to familial hemiplegic migraine and/or episodic ataxia type-2, derived from a gene present on chromosome 19p13.1-19p13.2; a gene encoding the α1 (ion-conducting) subunit of a P/Q-type voltage gated calcium channel. The present invention also provides access to and methods to study the genetic background and identify other subunits of the calcium channel subunit complexes and the proteins related therewith that are associated with the genetic factors and biological mechanisms that are related to various episodic neurological disorders such as FHM, EA-2, common migraine and others such as epilepsy which are related to cation channel dysfunction.

The sequence of the cDNA of the gene is highly related (≧90%) to a brain-specific rabbit and rat voltage gated P/Q-type calcium channel al subunit (Mori et al., 1991; Starr et al., 1991), and the open reading frame consists of 2261 amino acid residues. Northern blot analysis showed a brain-specific expression, especially in the cerebellum. Primary study of a cosmid contig harbouring the gene already indicated an exon distribution over at least 300 kb of genomic DNA. Recently, a neuronal Ca²⁺ α1A subunit gene was localized to chromosome 19p13.1-p13.2 by FISH analysis (Diriong et al, 1995). The gene symbol is CACNL1A4 and the al subunit is classified as a P/Q-type. No sequence data for the CACNL1A4 gene have been provided by Diriong or others, but the same localization (chromosome 19p13.1) and the identical classification (P/Q-type) suggests that the Ca²+ channel α1 subunit we have identified is very similar to CACNL1A4. No relation with migraine has been disclosed for CACNL1A4. The genomic structures of three other human Ca²+ channel α1 subunit genes (CACNL1A1, CACNL1A2 and CACNL1A3) have been published to date (Hogan et al, 1994; Soldatov, 1994; Yamada et al, 1995). Both CACNL1A1 and CACNL1A2 span about 150 kb and consist of 50 and 49 exons, respectively. The smaller CACNL1A3 gene is composed of 44 exons, distributed over 90 kb.

The present invention also provides an isolated and/or recombinant nucleic acid comprising alleles of the invented gene which contain mutations relevant to the occurence of migraine and other neurological disorders which are related to cation channel dysfunction. Such mutations are for example a mutation at codon 192 resulting in the replacement of arginine by glutamine (R192Q), and/or a mutation at codon 666 resulting in the replacement of threonine by methionine, and/or a mutation at codon 714 resulting in a replacement of valine by alanine and/or a mutation at codon 1811 resulting in a replacement of isoleucine by leucine, but also other mutations of alleles of said gene which bear relationships with cation channnel dysfunction.

The present invention also provides isolated and/or recombinant nucleic acid comprising alleles of said gene which contain a polymorphic CA-repeat sequence specific for various alleles of said gene. The present invention also provides isolated and/or recombinant nucleic acids comprising alleles of said gene which contain a CAG repeat.

The present invention also provides methods and tests (such as PCR, but also other tests to detect or amplify nucleic acids are known in the art) to detect, identify and localize or distinguish genes and alleles of such genes, or fragments thereof, encoding for proteins or α, β or χ sub-units of specific cerebral cation channels, more specifically the invented gene and its various alleles encoding the α1 subunit of a P/Q-type voltage gated calcium channel and the gene encoding the β2 sub-unit, which are involved in the primary pathogenesis of neurological disorders such as FHM, migraine, EA-2 and SCA6. With such methods and tests one can study abnormalities of said gene.

The invention also provides recombinant expression vectors comprising isolated and/or recombinant nucleic acid comprising alleles of said genes or fragments therof, and provides host cells or animals that comprise such vectors or that are otherwise transformed with an isolated and/or recombinant nucleic acid according to the invention.

The invention thus also provides a rationale and methods for the testing and the development of specific prophylactic medication for migraine and other episodic neurological, in particular brain, disorders, such as epilepsy, associated with cation channel dysfunction.

The invention for example provides cells or animals that comprise recombinant vectors that comprise variants of said genes or cells or animals that are transformed with said variants. Also, the invention provides means to identify naturally occuring variants of experimental animals with changes in said gene related to FHM, EA-2, SCA7, migraine or other neurological disorders associated with cation channel dysfunction. An example of such an animal is the tottering mouse, and its variants called leaner and rolling, described in the experimental part of the invention. The invention also provides cells or animals in which changes such as deletions or mutations in said gene have been introduced by recombinant nucleic acid techniques. All such cells or animals provided by the invention can be used to study the pathophysiology of FHM, EA-2, migraine or other neurological disorders associated with cation channel dysfunction, for example to test or develop specific medication for the treatment of said disorders.

The invention also provides proteins or peptides encoded by said genes, or fragments thereof, related with cation channel dysfunction, and detection of such proteins or peptides by antibodies directed against said proteins or peptides. Such antibodies can be of natural or synthetic origin, and can be produced by methods known in the art. Such proteins and antibodies and detection methods can be used to further in vitro or in vivo studies towards the pathophysiology of FHM, EA-2, migraine or other neurological disorders associated with cation channel dysfunction, in addition such proteins, antibodies and detection methods can also be used to diagnose or identify such disorders in patients or in experimental animals.

Experimental Procedures Subjects

Sixteen FHM patients were selected, including eight individuals from four unrelated chromosome 19-linked FHM families (NL-A, UK-B, USA-C (Ophoff et al, 1994), and USA-P (Elliot et al., 1995), two affected individuals from two small FHM families from Italy (Italy I & II) and six individuals with sporadic hemiplegic migraine (i.e. no other family member was shown to suffer from attacks of hemiplegic migraine). In families NL-A and USA-P cerebellar ataxia and/or nystagmus is associated with FHM. An additional set of four subjects from four unrelated EA-2 families linked to chromosome 19, was also included (CAN-25, -45, -191, -197. Fifty randomly collected individuals from the Dutch population (Smith et al., 1988) were used as a control to determine the allele frequencies of polymorphic sites.

Patients with migraine with or without aura were diagnosed according to the international Headache-Society (IHS) classification criteria. Patients attending the neurology outpatient clinic of Leiden University Medical Center, The Netherlands, and patients responding to calls in local newspapers or in the periodical of the Dutch Migraine Patients Association, were screened for a positive family history of migraine. Only families with migraine in at least two generations were asked to participate. Probands (n=36) and relatives (n=492) were personally examined and interviewed using semi-structured questionnaires. The questionnaire included questions about age at onset, frequency and duration of attacks, aura symptoms, premonitory signs and symptoms, triggers for attacks, medication, and additional headaches. When family members were not available for a personal interview, information on their migraine was collected by interviewing their relatives. Because of the low validity of diagnosing migraine auras through relatives, we only assessed the presence or absence of migraine headaches. Whenever possible, medical records were examined.

Genomic Structure

Ten different cosmids from the contig extending the invented gene, were subcloned separately in either M13 or pBlueScript KS vector. From each cosmid library at least 3×96 random clones with an average insert size of about 2 kb, were picked. Positive clones were identified by hybridization techniques and subsequently sequenced with vector-specific primers; intron-exon boundary sequences were completed using cDNA-based primers.

Mutation Analysis, DHPLC and SSCP

Genomic DNA was used as template to generate polymerase chain reaction (PCR) products for single-strand conformational polymorphism (SSCP) analysis and denaturing high-performance liquid chromatography (DHPLC). Amplifications were performed in standard conditions with primer pairs as listed in Table 1 or listed below. Except for the 5′ side of exon 6, primers were chosen to produce fragments that contained a single exon and at least 35 basepairs (including primer) of each flanking intron sequence. Amplification of exons 1 and 20 was performed producing two overlapping fragments and exon 19 was amplified into three overlapping fragments. In addition, the following markers;

D10S191 Primer sequence 1 (SEQ ID NO: 141) CTT TAA TTG CCC TGT CTT C

Primer sequence 2 (SEQ ID NO: 142) TTA ATT CGA CCA CTT CCC

D10S245 Primer sequence 1 (SEQ ID NO: 143) AGT GAG ACT CGT CTC TAA TG

Primer sequence 2 (SEO ID NO: 144) ACC TAC CTG AAT TCC TGA CC

DIOS89 Primer sequence 1 (SEQ ID NO: 145) AAC ACT AGT GAC ATT ATT TTC A

Primer sequence 2 (SEQ ID NO: 146) AGC TAG GCC TGA AGG CTT CT

DHPLC (Oefner et al., 1995; Hayward et al., 1996) was carried out on automated HPLC instrumentation. Crude PCR products, which had been subjected to an additional 3-minute 95° C. denaturing step followed by gradual reannealing from 95-65° C. over a period of 30 minutes prior to analysis, were eluted with a linear acetonitrile (9017-03, J. T. Baker, Phillipsburg, N.J., USA) gradient of 1.8% per minute at a flow-rate of 0.9 ml/min. The start- and end-points of the gradient were adjusted according to the size of the PCR products (Huber et al., 1995). The temperature required for successful resolution of heteroduplex molecules was determined empirically by injecting one PCR product of each exon at increasing mobile phase temperatures until a significant decrease in retention was observed.

For SSCP analysis, primary PCR products were labeled by incorporation of [α-³²P]dCTP in a second round of PCR. Samples were diluted and denatured in formamide buffer before electrophoresis. SSCP was carried out according to published protocols (Orita et al., 1989; Glavac et al., 1994). Digestion of several exons to yield products suitable for SSCP analysis.

Sequencing of PCR products was performed with an ABI 377 automated sequencing apparatus with cycle sequencing according to the manufacturer. Furthermore, PCR products were cloned in the TA vector (Invitrogen) and subjected to manual dideoxy sequence analysis (T7 Sequencing kit, Pharmacia Biotech.).

A total of 481 blood samples were collected from patients with migraine. Genomic DNA was isolated as described by Miller et al., 1988. The analyses of the highly informative microsatellite markers D19S391, D19S394, D19S221 and D19S226, D10S191, D10S248 and D10S89 were performed by PCR; primer sequences related to these markers are available through the human Genome Data Base (GDB).

The relative frequencies of marker alleles were estimated on the entire family material, with the relevant correction for genetic relationships between individuals (Boehnke, M, 1991) with the ILINK option of the I-INKAGE package, version 5.03 (Lathrop et al., 1985). The following marker order and recombination frequencies were used in the multipoint sib-pair analysis: D19S391-5%-D19S394-3%-D19S221-5%-D19S226. Affected-sib-pair analysis was performed using the MAPMAKER/SIBS software package, simultaneously including marker information for all four DNA markers (Kruglyak, 1995). Separate analyses were performed for migraine with aura, migraine without aura, and a combination of both. Allowance was made for dominance variance. When more than two affected sibs occurred in a single sibship, weighted scores were computed according to Suarez and Hodge (1979).

In a sib-pair analysis, the occurrence of parental marker alleles is compared among sibs. Normally, 25% of sib pairs share their marker alleles from both parents, 50% share one marker allele from one of their parents, while the remaining 25% share no parental allele. Deviations from this pattern towards increased sharing, and consistent with the constraints of Holmans's (1993) possible triangle, are explained as linkage (expressed as the maximum lod score MLS). Increased sharing of marker alleles thus indicate that the marker is located closely near a genetic risk factor. The relative-risk ratio for a sib (λ_(R)), defined as the ratio of the prevalence of a disease in sibs of affected individuals, divided by the prevalence of a disease in the population, can be calcutated (May et al., 1995). In other words: $\lambda_{p} = \frac{{Affected}\quad {risk}\quad {for}\quad {sib}\quad {of}\quad a\quad {proband}}{{Affection}\quad {risk}\quad {for}\quad {an}\quad {individual}\quad {in}\quad {the}\quad {general}\quad {population}}$

Results Genomic Structure

The combination of hybridization and PCR strategies resulted in a rapid assembly of the complete coding sequence of the human cDNA, with an open reading frame of 6783 nucleotides encoding 2261 amino acid residues (FIG. 4). The spatial distribution of the human Ca²+ channel expression was assayed in rhesus monkey tissues. Total RNA was isolated from several tissues, including various brain structures, and probed with a human cDNA fragment. The probe detected a major transcript of approximately 9.8 kb in cerebellum, cerebral cortex, thalamus and hypothalamus, whereas no transcript was detected in heart, kidney, liver or muscle. There was also no hybridization signal found in RNA preparations from mouse skin tissue or from human peripheral lymphocytes. In addition, an attempt to amplify parts of the cDNA from human peripheral lymphocytes failed.

Complete alignment between the cDNA and individual exon sequences was achieved, allowing the establishment of the exon-incron structure (Table 1). The reconstruction of the exon-intron structure of the CACNL1A4 gene revealed 47 exons ranging in size from 36 bp (exon 44) to 810 bp (exon 19). The exons are distributed over about 300 kb at genomic DNA level. The result shows that the first 10 exons are located in a region of about 150 kb covered by the first 5 cosmids of the contig indicating relatively large introns at 5′ side of the gene. Sequences (FIG. 1) were obtained of all exons including approximately 100 bp of flanking introns, except for intron 5 adjacent to exon 6. The forward primer of exon 6 harbours the splice junction and 3 bp of exon 6. Splice sites around all exons are compatible with consensus sequence with the exception of splice donor and acceptor of the first intron.

The cosmid conzig that yielded the initial Ca²+ channel gene exons was extended to cover more than 300 kb. Hybridization experiments showed that the first and last cosmids of the contig were positive for 3′- and 5′-end cDNA sequences, respectively, indicating a genomic distribution of the gene over at least 300 kb (FIG. 2). The cosmid contig has been placed into the LLNL physical map of chromosome 19 at band p13.1, between the markers D19S221 and D19S226 (FIG. 2). We identified a new polymorphic CA-repeat sequence (D19S1150) on the cosmid contig. Oligonucleotide primers (Table 1) flanking the repeat were synthesized and amplification was performed by PCR as described. Analysis of D19S1150 in 45 random individuals from the Dutch population revealed nine alleles with an observed heterozygosity of 0.82. This highly polymorphic marker is located within the gene and is therefore very useful in genetic analysis.

Mutation Analysis

Exons and flanking intron sequences, containing the complete coding region of CACNL1A4 and part of untranslated sequences, were screened for the presence of mutations by SSCP and DHPLC analysis in 20 individuals with either FHM or EA-2. Several synonymous nucleotide substitutions and polymorphisms were identified including a highly polymorphic (CAG)n-repeat in the 3′ untranslated region of exon 47 (Table 2). Of all polymorphisms only one was identified predicting an amino acid change, an alanine to threonine substitution at codon 454 (A454T).

Four different missense mutations were found in FHM patients of which one mutation was observed in two unrelated FHM affected individuals (Table 3). The mutations were shown to segregate with the disease within the families, and were not present in about 100 control chromosomes. A G-to-A transition was identified in family Italy-II at codon 192, resulting in a substitution of arginine to glutamine (R192Q) within the first voltage sensor domain (IS4). A second missense mutation occurs at codon 666, within the P-segment of the second repeat replacing a threonine residue for methione (T666M) in family USA-P. Two other mutations are located in the 6th transmembrane spanning segment of respectively repeat II and IV. The IIS6 mutation is a T-to-C transition at codon 714, resulting in a substitution of valine to alanine (V714A), identified in FHM family UK-B. The mutation in domain IVS6 is an A-to-C transversion at codon 1811 resulting in a substitution of isoleucine to leucine (I1811L). This I1811L mutation is found in family NL-A and family USA-C, two unrelated FHM families. Comparison of haplotypes in this region, including intragenic markers, did not reveal any evidence for a common founder of family NL-A and USA-C (data not shown). No mutation was found in FHM family Italy-I nor in the six sporadic hemiplegic migraine patients. In addition to missense mutations in FHM families, we also identified mutations in two out of four EA-2 families (Table 3). In EA-2 family CAN-191, a basepair deletion occurs in exxon 22 at nucleotide position 4073 causing a frameshift and a premature stop. The second EA-2 mutation is a transition of G-to-A of the first nucleotide of intron 24, predicted to leading to an aberrant splicing in family CAN-26. The invented gene also contains a CAG repeat, of which expansions have been found in patients with autosomal dominant cerebellar ataxia (SCA6). Hence FHM, EA-2 and SCA6 are alielic ion channel disorders and different mutations are associated with different clinical symptomatologies.

Our study patients with common migraine (with or without aura) included 36 independent multigenerational Dutch families. At least some data were available on 937 family members and 212 persons who “married-in” (spouses). Of these, 442 family members (247 affected) and 86 spouses (7 affected) were personally interviewed. The distribution of the different types of migraine among the 247 affected family members are as follows: 132 family members showed migraine without aura, 93 showed migraine with aura and 22 showed months-migraine, not fulfilling all critera by IHS. Among the 7 affected spouses these figures were 4, 1 and 2, respectively. We scored the parental transmission of migraine in the 36 families (Tabel 4) to investigate if an additional X-linked dominant or mitachondrial aene was involved. An approximately 2.5:1 preponderance of females among the migraine sufferers was noted, which remained in the affected offspring. Affected fathers were found to transmit migraine to their sons in 21 cases, making X-linked dominant or mitochondrial inheritance highly unlikely.

The genetic analysis included 204 potentially affected sib pairs; after correction for more than one sib pair in a single sibship the total number of sib pairs was 108. Affected-sib-pair analysis was performed for sib pairs who were both affected with any form of migraine and, in separate analyses, for sib pairs who where both suffering from either migraine with aura or migraine without aura. The informativeness of the region between the markers D19S391, D19S394, D19S221 and D19S226 varied between 82% and 96%. The combined analysis of migraine with and without aura resulted in a maximum multipoint lod score of 1.69 (p≈0.005) with marker D19S226. For migraine with aura the maximum multipoint lod score was 1.29 corresponding with p≈0.013 with marker D19S394. The maximum lod score for migraine without aura was not significant (MLS <0.25)(data not shown). The relative risk ratio for a sib to suffer from migraine with aura (λ_(p)), defined as the increase in risk of the trait attributable to the 19p13 locus, varied between λ_(R)=1.5 (for marker D19S394) and λ_(R)=2.4 (for marker D19S226). When combining migraine with and without aura, λ_(R) was 1.25. In a selected portion of 36 Dutch families with migraine with aura and without aura, affected sib-pair analysis was performed for sib pairs who were affected with any form of migraine. The following markers, flanking the β2(CACNB2) calcium channel subunit gene on chromosome 10p12, were tested: D108191, D1OS246 and D10S89. For the combined phenotype (migraine with and without aura) a maximum pultipoint iod score of 0,95 (p<0,01) was obtained with marker; D10S191. This result gives independent evidence for a role of the P/Q type Ca²⁺ channel in migraine and other neurological disorders.

Discussion

The genomic structure of the exemplified invented gene revealed 47 exons distributed over about 300 kb (Table 1; FIG. 1). A comparison of the gene structure to already known Ca²⁺ channel al subunit genes (CACNL1A1, CACNL1A2, and CACNL1A3) (Soldatov, 1994; Yamada et al., 1995; Hogan et al., 1995), reveals a similar number of exons (50, 49, and 44 respectively) but a larger genomic span (300 kb vs 90-150 kb). Remarkebly, all splice sites are according to consensus sequence except for intron 1. Splice donor as well as splice acceptor of the first intron do not contain the expected gt . . . ag intron sequence. An incorrect CDNA sequence is unlikely because the cDNA sequence containing the junction of the first two exons is identical to rabbit and rat sequence. Sequences corresponding to splice donor and acceptor are present in exon 1 and 2, suggesting an additional (yet unidentified) exon in the first intron encompassing part of sequences of exon 1 and exon 2.

To test the possible involvement of the invented gene relating to the CA²+-channel sub-unit in migraine FHM, SCA6 and EA-2, we performed a mutation analysis by DHPLC and SSCP and found several alterations (For example Table 2 & 3). Only one missense variation was observed also present in 2% of the normal controls (Table 2). This polymorphism is a alanine to threonine substitution at codon 454 (A454T), located in the intracellular loop between IS6 and IIS1 (FIG. 2). This region contains a conserved alpha interaction domain (AID) that binds subunits (De Waard et al., 1996). However, A454T is located outside the AID consensus sequence and is not likely to be involved.

The identification of two mutations that disrupt the predicted translation product of the invented gene in two unrelated EA-2 patients and the segregation of these mutations with the episodic ataxia phenotype in their families provide strong evidence that the invented gene is the EA-2 gene. A basepair deletion leads to a frame-shift in the putative translation product and encounters a stop codon in the next exon. The frame-shift in this EA-2 family is predicted to yield a calcium channel al subunit polypeptide consisting of repeat I and II, and a small portion of repeat III (IIIS1). The G-to-A transition of the first nucleotide of intron 24 is affecting the nearly invariant GT dinucleotide of the intronic 5′ splice junction. The brain-specific expression of the exemplified invented gene makes it extremely difficult to test the hypothesis that this mutation produces aberrantly spliced RNAs by retaining the intron or utilizing other cryptic 5′ splice sites.

The frameshift and splice site mutations in EA-2 may suggest a dominant negative effect of the truncated proteins by overruling the (corresponding) intact al subunits.

No mutations were found in the remaining EA-2 families (CAN-25 and -197). The use of two independent techniques for mutation screening (DHPLC and SSCP) makes it unlikely that we missed a heterozygote PCR product. Mutations in the promoter region or in intron sequences, resulting in aberrant splicing, may have been the cause of EA-2 in these families. We could also have missed a mutation around the splice acceptor site of intron 5, covered by the forward primer of exon 6. However, larger deletions of e.g. complete exons with flanking intron sequence will disturb the predicted translation product, like the ΔC₄₀₇₃ and splice site mutation do, but this is not detectable by a PCR-based screening method but can be seen Southern blot analysis instead.

Four different missense mutations were identified in five unrelated FHM families. These mutations all segregate with FHM within a family and are not observed in over 100 normal chromosomes. The first missense mutation that we describe in the exemplified invented gene occurs in the IS4 domain of the al subunit (Table 3; FIG. 3). The S4 domains are postulated to be voltage sensors because they have an unusual pattern of positively charged residues at every third or fourth position separated by hydrophobic residues (Tanabe et al., 1987). In calcium channels the positively charged amino acid is an arginine residue (Stea et al., 1995). The mutation in FHM family Italy-II predicts a substitution of the first arginine in the IS4 segment with a neutral, non-polar alutamine (R192Q). The change of the net positive charge of this conserved region of the protein may influence correct functioning of the voltage sensor.

The second missense mutation in FHM family USA-P occurs in the P-segment of the second transmembrane repeat. A C-to-T transition predicts substitution of a threonine residue with methionine at codon 666 (T666M). Various observations have shown that P-segments, the hairpin between S5 and S6 that spans only the outer part of the transmembrane region, form the ion-selectivity filter of the pore and binding sites for toxins (Guy and Durell (1996). Alignment of protein sequence of different P-segments indicating that some residues occur in many different channel genes (Guy and Durell, 1996). The T666M substitution alters one of the conserved residues in the P-segment. It is conceivable that an alteration of a P-segment affects the ion-selectivity or toxin binding of a channel gene.

The remaining two missense mutations identified in FHM families alter the S6 segment of the second and the fourth repeat. A valine to alanine substitution in FHM family UK-B is found in domain IIS6 at codon 714 (V714A). Domain IVS6 is mutated in two unrelated FHM families (NL-A and USA-C), predicting a isoleucine to leucine substitution at codon 1811 (I1811L). The V714A and I1811L missense mutations do not really change the neutral-polar nature of the amino acid residues. However, both S6 mutations are located nearly at the same residue at the intracellular site of the segment and are conserved in all calcium channel al subunit genes. In addition, the A-to-C transversion leading the I1811L substitution occurred in two unrelated FHM families on different haplotypes indicating recurrent mutations rather than a founder effect. Although the exact function of the S6 domains are not known, these data strongly suggest that mutations in IIS6 and IVS6 result in FHM.

The I1811L mutation is present in two FHM families of which one (NL-A) also displays a cerebellar atrophy in (some) affected family members. The presence of cerebellar atrophy in FHM families has been reported in about 40% of chromosome 19-linked FHM families, whereas none of the unlinked families was found to have cerebellar atrophy (Terwindt et al., 1996).

The I1811L mutation excludes the possibility of allelic mutations in FHM and FHM with cerebellar atrophy. However, it is likely that FHM or FHM with cerebellar atrophy are the result of pleiotropic expression of a single defective gene.

No mutation was found in a small Italian FHM family (Italy-I). Apart from the possibilities discussed for EA-2, it should be noted that linkage to 19p13 was only suggested but never proved with significant lod scores (M. Ferrari, personal knowledge).

The four missense mutations identified indicate a mechanism for FHM in which both alleles of the α1 subunit are expressed, one harbouring an amino acid substitution which affects the function of this calcium channel α1 subunit by reducing or enhancing the electrical excitability. The relationship of FHM and other types of migraine makes it highly rewarding to investigate the involvement of the only missense variant observed (A454T) (Table 2), and to continue the search for other variants of the exemplified invented gene specific for common types of migraine.

The mutations in EA-2 and FHM demonstrate among others that the brain specific calcium channel gene CACNL1A4 is responsible for both EA-2 and FHM, and is also involved in the primary pathogenesis of the more common forms of migraine. We conducted the common migraine study in an independent sample of 36 extended Dutch families, with migraine with aura and migraine without aura. We found significant increased sharing of the marker alleles in sibs with migraine with aura (MLS=1.29 corresponding with p≈0.013). Although no such increased sharing was found for migraine without aura, a combined analysis for both migraine types resulted in an even more significant increased sharing (MLS=1.69 corresponding with p≈0.005). These results clearly indicate the involvement of the calcium cSIA-subunit gene region on 19p13 in both migraine with and without aura; the contribution to migraine with aura, however, seems strongest.

The positive findings in our study clearly demonstrate an involvement of the FHM locus region in non-hemiplegic familial migraine, notably in migraine with aura. The P/Q-type calcium channel α_(1A)-subunit gene on chromosome 19p13 may be an “aura-gene” and is involved in both FHM and migraine with aura, but not in migraine without aura. This however, seems unlikely since an increased sharing of marker alleles was also found when we combined the results for migraine with and without aura. Furthermore, the increase in sharing was stronger than expected to be only due to the contribution of migraine with aura. An alternative explanation is that the gene is involved in both types of migraine, but in migraine without aura there is an additional strong effect of other, possibly environmental factors, thereby reducing the penetrance.

The latter view is also supported by the results obtained from calculating the relative risk ratios (λ_(R)) for sibs from affected individuals to also have migraine. The relative risk ratio for a sib to suffer from migraine with aura was λ_(R)=2.4. When combining migraine with and without aura, λ_(R) was 1.25. In a population-based study the relative risk for first degree relatives of probands with migraine with aura to also have migraine with aura was λ_(R)=3.8. Because of the female preponderance among migraine patients, X-linked dominant or mitochondrial inheritance has been suggested to be involved in familial migraine. Although a predominant maternal inheritance pattern was noted in our families, X-linked dominant or mitochondrial inheritance were found to be highly unlikely because affected fathers transmit migraine to their sons. Furthermore, the predominant maternal inheritance can be explained by the female preponderance among the migraine patients.

We conclude that the well-established genetic contribution to the etiology of migraine is partly, but not entirely, due to genetic factors located in the chromosomal region of the P/Q-type calcium channel α_(1A)-subunit gene. Further analysis of the cerebral distribution and function of this calcium channel, as well as of the “mutated channels”, will help to unravel the pathogenetic pathway of migraine. It may also contribute to a better understanding of the mechanisms involved in related disorders such as episodic ataxia type-2, autosomal dominant cerebellar ataxia (SCA6), cerebral atrophy, and epilepsy, which all have been found to be associated with mutations in this gene. Study of FHM, EA-2 mutants and variants such as the A454T variant expressed in vitro or in mouse or other experimental animal models will ultimately lead to better understanding of the diseases, their cellular mechanisms, and the clinical relationship between FHM, EA-2, migraine, and other episodic neurological disorders such as epilepsy, and will provide rationales for the development of prophylactic therapy.

Localization and identification of the mouse gene related to the neurological mouse mutations tottering, leaning and rolling.

The tottering (tg) mutation arose spontaneously in the DBA inbred strain, and has been back-crossed into a C57BL/6J (B6) inbred strain for at least 30 generations. The genome of the tg mouse therefore is of B6 origin except for a small region around the tg gene on chromosome 8. Interestingly, the chromosome 8 region in mouse has synteny with the human chromosome 19p13.1, in which the human calcium channel alphal subunit has been identified. We therefore consider the tg locus as a possible site of the mouse homologue of the human calcium channel gene.

To determine the exact localization of the mouse homologue, PCR was carried out with primers based on human cDNA sequence selected from FIG. 1 and mouse genomic DNA aE template. In human, primers were known to be located in different flanking exons. POR amplification on human DNA yielded a 1.5kb fragment.

Forward primer (SEQ ID NO: 45)5′-caa cat cat gct ttc ctg cc-3′

Reversed primer (SEQ ID NO: 46)5′-atg atg acg gcg aca aag ag-3′

Amplification on mouse DNA yielded a 750-bp fragment. The fragment mainly consists of intronic sequences. SSCP analysis revealed several polymorphisms in the different inbred strains (each strain a specific pattern). Analysis of amplified product of the tg/tg (homozygote) and tg/+(heterozygote) mice demonstrated a DBA specific signal in the tg/tg mouse, and a heterozygous pattern of DBA and B6 inbred strains in the heterozygous tg/+mouse. These results show that the mouse homologue of the human calcium channel alphal subunit is located within the mouse tottering interval on chromosome 8.

In conclusion: the phenotypic characteristics of the tg mouse (tg/tg and tg/+) suggest involvement of ion-channels in the tg-etiology. The localization of the mouse homologue of the human calcium gene within the tottering interval show that a tottering phenotype in mouse is caused by a mutation in the mouse homologue of the CACNL1A4 gene. With various variants of the tottering mouse (the Jackson Laboratory, Bar Habor, Me., USA), such as the leaner and rolling varieties, such mutations in the mouse homologue of the CACNL1A4 gene can be found, clearly demonstrating that the gene is related to a variety of episodic neurologic disorders and using this genetic information one can engage in a variety of pathofysiological studies, as for example indicated below.

The tg mutation arose spontaneously in the DBA/2 inbred strain. tg/tg homozygotes are characterized by a wobbly gait affecting the hindquarters in particular, which begins at about 3 to 4 weeks of age, and by intermittent spontaneous seizures which resemble human epileptic absence seizures. The central nervous system of the mice appears normal by light microscopy. There is no discernible cerebellar hypoplasia. In fluorescent histochemistry studies tg/tg mice show a marked increase in number of noradrenergic fibers in the terminal fields innervated by locus ceruleus axons, the hippocampus, cerebellum, and dorsal lateral geniculate. Treatment of neonatal tg/tg mice with 6-hydroxydopamine, which selectively causes degeneration of distal noradrenergic axons from the locus ceruleus, almost completely abolishes the ataxic and seizure symptoms.

The leaner mutation of the tottering mouse arose spontaneously in the AKR/J strain. Homozygotes are recognized at 8 to 10 days of age by ataxia, stiffness, and retarded motor activity. Adults are characterized by instability of the trunk, and hypertonia of trunk and limb muscles. The cerebellum is reduced in size, particularly in the anterior region, in tg<la>/tg<la>mice, as is the case with a certain number of FHM patients. There is loss of granule cells beginning at 10 days of age and loss of Purkinje and Golgi cells beginning after 1 month. Cell loss later slows but continues throughout life. Granule and Purkinje cells are more severely affected than Golgi cells and the anterior folia more severely affected than other parts of the cerebellum. The cerebellum of tg<la>/tg mice shows shrinkage and degenerative changes of the Purkinje cells. The loss in cerebellar volume in tg<la>/tg and in tg/tg mice is specific to the molecular layer, with no change in volume of the granule cell layer or the white matter layer. Allelism of aleaner with tottering was shown in complementation and linkage tests.

A third variety of the tottering mouse is (tg<rol>) called the rolling Nagoya. Found among descendants of a cross between the SIII and C57BL/6 strains, the tg<rol>mutation apparently occurred in the SIII strain. Homozygotes show poor motor coordination of hindlimbs that may lead to falling and rolling, and sometimes show stiffness of the hindlimbs and tail. No seizures have been observed. Symptoms are recognizable at 10 to 14 days old. They appear a little earlier than those of tg/tg mice and are somewhat more severe. The cerebellum is grossly normal until 10 days of age, but after that grows more slowly than normal. The size of the anterior part of the central lobe of the cerebellum is reduced with reduction in the numbers of granule, basket, and stellate cells but normal numbers of Purkinje cells. There is a reduced concentration of glutamate and an increased concentration of glycine and taurine in the cerebellum and decreased activity of tyrosine hydroxylase in the cerebellum and other areas.

LEGENDS TO FIGURES

FIG. 1

Nucleic acid sequences of 47 exons and flanking intron sequences containing the complete coding region of the invented gene and part of untranslated sequences SEQ ID NO: 41-SEQ ID NO: 42.

FIG. 2

Genetic map, cosmid contig and global exon distribution of the invented gene om chromosome 19p13.1. The cosmid contog is shown with EcoRI restriction sites, available via Lawrence Livermore National Laboratory; exon positions are indicated schematically, regardless of exon or intron sizes (Table 1). D19S1150 is a highly polymorpmic intragenic (Ca)_(n-repeat).

FIG. 3

Membrane topology of α1 subunit of the P/Q-type Ca²⁺-channel. The location and amino acid substitutions are indicated for mutations that cause FHM or EA-2.

FIG. 4

The coding sequence SEQ ID NO:43 of human cDNA of the invented gene with an open reading frame encoding 2261 amino acid residues SEQ ID NO: 44.

REFERENCES

1. Browne, D. L., Gancher S. T, Nutt, J. G., Brunt, E. R., Smith E. A., Kramer P., and Litt M. (1994). Episodic ataxia/myokymia syndrome is associated with point mutations in the human potassium channel gene, KCNA1. Nat. Genet. 8: 136-140.

2. Diriong S., Lory P., Williams M. E., Ellis S. B., Harpold M. M., and Taviaux S. (1995). Chromosomal localization of the human genes for α1A, α1B, and α1E voltage-dependent Ca²+ channel subunits. Genomics 30: 605-609.

3. Headache Classification Committee (HCC) of the International Headache Society (1988). Classification and diagnostic criteria for headache disorders, cranial neuralgias and facial pain Cephalalgia 8: 19-28.

4. Hogan, K.,Powers, P. A., and Gregg, R. G. (1994). Cloning of the human skeletal muscle alpha 1 subunit of the dihydropyridine-sensitive L-type calcium channel (CACNL1A3). Genomics 24: 608-609.

5. Joutel A., Bousser M-G., Biousse V., Labauge P., Chabriat H., Nibbio A.,Maciazek J., Meyer B., Bach M-A., Weissenbach J., Lathrop G. M., and Tournier-Lasserve E. (1993). A gene for familial hemiplegic migraine maps to chromosome 19. Nature Genet. 5: 40-45.

6. Joutel A., Ducros A., Vahedi K., Labauge P., Delrieu O., Pinsard N., Mancini J., Ponsat G., Gaoftiere F., Gasant J. L., Maziaceck J. Weissenbach J., Bousser M. G., and Tournier-Lasserve E. (1994). Genetic heterogeneity of familial hemiplegic migraine. Am. J. Hum. Genet. 55: 1166-1172.

7. Hayward-Lester, A., Chilton, B. S., Underhill, P. A., Oefner, P. J., Doris, P. A. (1996). Quantification of specific nucleic acids, regulated RNA processing and genomic polymorphisms using reversed-phase HPLC.

In: F. Ferr (Ed.), Gene Quantification, Birkhuser Verlag, Basel, Switzerland.

8. Huber, C. G., Oefner, P. J., Bonn, G. K. (1995) Rapid and accurate sizing of DNA fragments by ion-pair chromatography on alkylated nonporous poly(styrene-divinylbenzene) particles. Anal. Chem., 67, 578-585.

9. Kramer P. L., Yue Q., Gancher S. T., Nutt J. G., Baloh R., Smith E., Browne D., Bussey K., Lovrien E., Nelson S, and Litt M. (1995). A locus for the nystagmus-associated form of episodic ataxia maps to an 11-cM region on chromosome 19p. Am. J. Hum. Genet. 57: 182-185.

10. May A, Ophoff R. A., Terwindt G. M., Urban C., Van Eijk R., Haan J., Diener H. C., Lindhout D., Frants R. R., Sandkuiji L. A., and Ferrari M. D. (1995). Familial hemiplegic migraine locus on 19p13 is involved in the common forms of migraine with and without aura. Hum. Genet. 96: 604-608.

11. Mori Y, Friedrich T., Kim M. S., Mikami A., Nakai J., Ruth P., Bosse E., Hofmann F., Flockerzi V., Furuichi T., Mikoshiba K. Imoto K., Tanabe T., and Numa S. (1991). Primary structure and functional expression from complementary DNA of a brain calcium channel. Nature 350: 398-402.

12. Oefner, P. J., Underhill, P. A. (1995) Comparative DNA sequencing by denaturing high-performance liquid chromatography (DHPLC). Am. J. Hum. Genet. 57 [Suppl.], A266.

13. Ophoff R. A., Van Eijk R., Sandkuijl L. A., Terwindt G. M., Grubben C. P. M., Haan J., Lindhout D., Ferrari M. D., and Frants R. R. (1994). Genetic heterogeneity of familial hemiplegic migraine. Genomics 22: 21-26.

14. Orita, M., Suzuki, Y., Sekiya, T., and Hayashi, K. (1989). Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5: 874-879.

15. Ptacek L. J., George A. L., Griggs R. C., Tawil R., Kallen R. G., Barchi R. L., Robertson M., and Leppert M. F. (1991). IdentifLication of a mutation in the gene causing hyperkalemic periodic paralysis. Cell 67: 1021-1027.

16. Ptacek L. J., Tawil R., Griggs R. C., Engel A. G., Layzer R. B., Kwiecinski H., McManis P. G., Santiago L., Moore M., Fouad G., Bradley P., and Leppert M. F. (1994). Dihydropyridine receptor mutations cause hypokalemic periodic paralysis. Cell 77: 863-868.

17. Ravnik-Glavac, M., Glavac D., and Dean, M. (1994). Sensitivity of single-strand conformation polymorphism and heteroduplex method for mutation detection in the cystic fibrosis gene. Hum. Mol. Genet. 3: 801-807.

18. Russell, M. B., Rasmussen B K., Thorvaldsen P., and Olesen J. (1995). Prevalence and sex-ratio of the subtypes of migraine. Int. J Epidemiol. 24: 612-618.

19. Starr T. V. B., Prystay W., and Snutch T. (1991). Primary structure of a calcium channel that is highly expressed in the rat cerebellum. Proc. Natl. Acad. Sci. 88: 5621-5625.

20. Soldatov N. M. (1994) Genomic structure of Human L-type Ca²+ channel. Genomics 22: 77-87.

21. Teh B. T., Silburn P., Lindblad K., Betz R., Boyle R., Schalling M., and Larsson C. (1995). Familial periodic cerebellar ataxia without myokymia maps to a 19-cM region on 19p13. Am. J. Hum. Genet. 56: 1443-1449.

22. Terwindt G. M., Ophoff R. A., Haan J., Frants R. R., and Ferrari M. D. (1996). Familial hemiplegic migraine: a clinical comparison of families linked and unlinked to chromosome 19. Cephalagia 16: 153-155.

23. Von Brederlow, B., Hahn, A. F., Koopman, W. J., Ebers, G. C., and Bulman, D. (1995). Mapping the gene for acetozolamide responsive hereditary paroxysmal cerebellar ataxia to chromosome 19p. Hum. Mol. Genet. 2: 279-284.

24. Yamada Y., Masuda K., Li Q., Ihara Y, Kubota A., Miura T., Nakamura K., Fujii Y., Seino S., and Seino Y. (1995) The structures of the Human Calcium channel α1 subunit (CACNL1A2) and β-subunit (CACNLB3) genes. Genomics 27: 312-319.

TABLE 1 Exon/intron organization of the human invented gene and exon-specific primer pairs Exon cDNA Size Domain Cosmid(s) Primer Forward Primer Reversed Size 1 UTR-568 500 25960/30151 tct ccg cag tcg tag ctc ca 53 ggt tgt aga gtg cca tgg tc 87 320 cgc aaa gga tgt aca agc ag 54 att ccc aag cct cca ggg tag 88 370 2 569-674 106 I S1     30151 cac ctc caa cac cct tct tt 55 tct gtg ccc tgc tcc act c 89 240 3 675-814 140 I S2, I S3     30151 acg ctg acc ttg cct tct ct 56 caa cca aaa gcc tcg taa tc 90 230 4 815-906 92 I S3, I S4 28913 aaa acc cac cct ctg ttc tc 57 ttg tca ggg tcg gaa act ca 91 160 5  907-1059 153 28913/27415 ctt ggt ggc ggg gtt t 58 ctg cct aat cct ccc aag ag 92 290 6 1060-1253 194     27415 tcc ctt ccc ttt tgt aga tg 59 gtg ggg ctg tgt tgt ctt t 93 350 7 1254-1357 104 I S6     27415 gac aga gcc aca aga gaa cc 60 agc aaa gag gag tga gtg gg 44 250 8 1358-1473 116 34077/27415 ata ctc tgg ctt ttc tat gc 61 gca tga ctc tct ttg tac tc 95 230 9 1474-1530 57 34077 gca gag aat ggg ggt gg 62 ctg agg tgg gtt tag agc ac 96 180 10 1531-1623 93 34077 ggg taa cgt ctt ttt ctc ttg c 63 atg tct ctt ggg cga tag gt 97 200 11 1624-1833 210 II S1 16894/32236 att tct tct gaa gga aca gc 64 gga ggg atc agg gag ttc gc 98 310 12 1834-1946 113 II S2, II S3 16894 caa gcc taa cct cct ctc tg 65 tca ttc cag gca aga gct g 99 200 13 1947-2051 105 II S3, II S4 16894 att tgg agg gag gag ttt gg 66 tca ctt tcc caa ctt tct gg 100 310 14 2052-2191 140 II S4, II S5 16894 cag aaa gtt ggg aaa gta gc 67 ttg aat tcc tgt gaa gga c 101 250 15 2192-2264 73 16894 ctt gga gat gag ata ctg agc 68 cag gca ctt tca tct gtg ac 102 200 16 2265-2382 118 II S6 16894 tcc aca gct gca tct cca ag 69 acc ctc cct tga gcc cc 103 270 17 2383-2450 68 II S6 16894 cag tgg ttg ctt ttc ctg ac 70 ttg cca gag aaa cat tct cc 104 130 18 2451-2557 107 16894 tga aca aag att cca cgt gc 71 ttc agg agc cag ggt agc atc 105 170 19 2558-3367 810 16894 tag caa tgc tct aag tcc tc 72 tgt ttc ctg agg aag tcc tc 106 320 cgc agg aga acc gca aca a 73 gcg atg acg tcg atg ctc 107 450 gc agc agg gag agc cgc agc 74 tac cgt cat tct gcg gat tc 108 300 20 3368-3831 464 16894 ggt tct ttt tca ttc act tgc 75 ttt cct ggc agt ctt agc tc 109 430 gag aat agc ctt atc gtc ac 76 cag tga tgt gag agc aga 110 200 21 3832-3973 142 16894/34275 tgg gaa att gtg gag gga gc 77 tga ctt ccg cca ccc tgg tg 111 250 22 3974-4103 130 III S1 16894/34275 agc ctg tgg tct gag tgg ac 78 tag gaa ggg gtg tgc tct gtc 112 210 23 4104-4163 60 III S2, III S3 16894/34275 atc cac tgc tct ctt gct tt 79 gtg gtt ctc act tat aat ctg 113 170 24 4164-4270 107 III S3     34275 tgg cct cat tgg ctt ccc tgc 80 aag agg aaa ccc ttg cga ag 114 250 25 4271-4370 100 III S4     34275 cta ccc aac ctg acc tct gc 81 aca tga taa ccc tga cag tc 115 220 26 4371-4531 161 III S5     34275 ctc atg ctc tct gtc aac tc 82 tgg ttc caa tgg gaa tgt gc 116 250 27 4532-4669 138     34275 ctg ctt ccc aag cag tct ag 83 tcc tgg ata gat ttc cag tc 117 300 28 4670-4871 202 III S6     34275 agt ttt taa agg aca gat gg 84 ttt ccc tgc ccc att cct ttg c 118 280 29 4872-5036 165 IV S1     34275 ctc tgc cgc tct cac cac tg 85 ttt atc agg tag agg cag g 119 250 30 5037-5147 111 IV S1, IV S2     34275 ttc caa gcc cat agc tgt agc 86 tga ccc tgc tac tcc tgc ttc 120 180 31 5148-5231 84 IV S3 15496 act gtg cct cta aca tgc ac 121 aag tgc tgg ctc aag cag 138 250 32 5232-5348 117 IV S4 15496 tct gtg agt ggt gac agc tc 122 gtc acc tgt ctt ctc agc 139 240 33 5349-5414 66 IV S5 15496 tgg aag gac tct ggc acg tg 123 gga ggc tct ggg aac ctt ag 140 250 34 5415-5530 116 15496 aga agc cac tgg agg aat ggc 124 att atc aga gca ggt ccc ctt c 141 250 35 5531-5681 151 IV S6 15496 tcc gag tct ctg att tct cc 125 aga cgg ccc tca cag tgt c 142 210 36 5682-5809 128 IV S6 15496 ttc att ccc tcg gtc tct gc 126 ctg act gaa cct gtg aga c 143 350 37 5810-5906 97 15496 tgt gaa ccc att gcc tgc a 127 tgg gaa tga ctg cgc ttg c 144 200 38 5907-6012 106 15496 atg cct ggg aat gac tgc 128 tgt cac gcc tgt ctg tgc 145 200 39 6013-6120 108 15496 tga cac cca ggc agg cag 129 tct gtc ctg gtg gat tgg atc 146 200 40 6121-6221 101 15496 ttg gtg agc tca ccg tgt 130 ttc ccg tgg tga cat gca agc 147 200 41 6222-6331 110 15496 gtc cac aca ctg ctc tct gc 131 aca ctc cac ctc cct ggc 148 320 42 6332-6470 139 15496 gcc agg gag gtg gag tgt 132 ggt tcc ttc cac cgc aac 149 550 43 6471-6584 114 15496/30762 caa ctc ccc aat ggc tc 133 cct acc cag tgc aga gtg agg 150 350 44 6585-6620 36 15496/30762 tct gtg tgc acc atc cca tg 134 aag gat tgg gct cca tgg ag 151 200 45 6621-6807 187 15496/30762 gtt ggt gct agc tgc tga c 135 ctt tct tct tcc tta gtg tc 152 330 46 6808-7061 254 15496/30762 gtg tgc tgt ctg acc ctc ac 136 agc ctg ggg tca ctt gca gc 153 320 47 7062-UTR ≧350    /30762 cct tgg ttt caa ttt tcg tgt ag 137 tgg ggc ctg ggt acc tcc ta 154 280 Note. Sizes of exons and PCR products are given in basepairs; domains of protein are indicated according to Stea et al., 1995. ( ) The sequence identification number (SEQ ID NO:) is in parenthesis immediately following each sequence.

TABLE 2 Polymorphisms in coding sequence of the invented gene Location Nucleotide change Frequency Consequence exon 4 nt 854 G - A Thr₁₉₃ 0.02 — exon 6 nt 1151 A - G Glu₂₉₂ 0.07 — exon 8 nt 1457 G - A Glu₃₉₄ 0.38 — exon 11 nt 1635 G - A Ala₄₅₄ 0.02 Ala₄₅₄ - Thr (A454T) exon 16 nt 2369 G - A Thr₆₉₈ 0.12 — exon 19 nt 3029 G - A Glu₉₁₈ 0.07 — exon 23 nt 4142 T - C Phe₁₂₈₉ 0.22 — exon 46 nt 6938 T - C His_(222′) 0.46 — exon 47 nt 7213 (CAG)_(n) 3′UTR # — Note Frequency as observed in 100 control chromosome: # Seven alleles of (CAG)_(n) were observed in the range between n = 4 to n = 14, with a heterozygosity value of 0.75.

TABLE 3 Mutations of the invented gene in families with FHM or EA-2 Disease Family Location Domain Nucleotide change Consequence FHM It-II exon 4 I S4 nt 850 G - A Arg_(192 - Gln) R192Q (gain of Sfcl site) FHM US-P exon 16 P-segment nt 2272 C - T Thr₆₆₆ - Met T666M FHM UK-B exon 17 II S6 nt 2416 T - C Val₂₁₄ - Ala V714A (gain of Bovl site) FHM NL-A/US-C exon 36 IV S6 nt 5706 A - C Ile₁₈₁ - Leu I1811L (gain of MnII site) EA-2 CAN-191 exon 22 III S1 nt 4073 deletion C frameshift STOP₁₂₉₄ (loss of NiaIV site) EA-2 CAN-26 intron 24 space site nt 4270-1 G - A AC/gt - AC/at aberrant (loss of BsaAl site) splicing

TABLE 4 Parental transmission of migraine for 36 unrelated Dutch families. affected parents N offspring N N (%) ratio* heathy father x migraine 51 daughters 72 48 (66.7%) 2.3:1 mother sons 72 21 (29.2%) migraine father x healthy daughters 26 17 (65.4%) 2.5:1 mother 18 sons 15  4 (26.7%) *ratio of proportion affected sons/proportion affected daughters

146 1 953 DNA human 1 tttttttacg ttctcttttt tttcgagtgg tgactggatg ctgattcttc ctcgtatttt 60 tgctgcttct ctctccctcc cctccttccc gggcccgggc ccgccccgca ccctccttcc 120 gcccctcctt ctccggggtc agccaggaag atgtcccgag ctgctatccc cggctcggcc 180 cgggcagccg ccttctgagc ccccgacccg agcgccgagc cgccgcgcga tgggctgggc 240 cgtggagcgt ctccgcagtc gtagctccag ccgccgcgct cccagccccg gcagcctcag 300 catcagcggc ggcggcggcg gcggcggcgt cttccgcatc gttcgccgca gcgtaaccgg 360 agccctttgc tctttgcaga atggcccgct tcggagacga gatgccggcc cgctacgggg 420 gaggaggctc cggggcagcc gccggggtgg tcgtgggcag cggaggcggg cgaggagccg 480 ggggcagccg gcagggcggg cagcccgggg cgcaaaggat gtacaagcag tcaatggcgc 540 agagagcgcg gaccatggca ctctacaacc ccatccccgt ccgacagaac tgcctcacgg 600 ttaaccggtc tctcttcctc ttcagcgaag acaacgtggt gagaaaatac gccaaaagat 660 caccgaatgg ccatatcctt ttgcccgaac cccagcagca gctgcgcctc cccctcctcc 720 ctccgcctcc cctcttccag gctgggagag agacccgggg gttgatggga ggtggggagg 780 aggggggtct tccaggggct gggagagggg gcaccgggag gagtgtgaaa gaatctctcc 840 accccgagct gggttgagct accctggagg cttgggaatg ggtttttcgg gggctggggg 900 ccggccagcc ggagagtgga tccttcccaa ggaccgactc tagaatgaga tct 953 2 527 DNA human 2 gatctttycc actggggtca gtgggggtgg gtgcacctcc aacacccttc ttttctttga 60 acaagatttt tccttaattc cccaatactc cctttgaata tatgatttta gccaccatca 120 tagcgaattg catcgtcctc gcactggagc agcatctgcc tgatgatgac aagaccccga 180 tgtctgaacg gctggtgagt gatgtctttt ctcagggtct tctccttggc tttagcagga 240 cattaatttt tgggggagtg gagcagggca cagaggaggc tctcagtcct ggagcccaga 300 gccagatcat gggaagccta aatttccttt tcattttttc ttgaaccaga gtctcgctct 360 gtcacccagg ctggagtgca gtggttcagt catagctcac tgcagcctcc acctcctggg 420 ctcaagccat cctcccactg cagcctcctg agtagcaggg actaacaggt gccaccatgc 480 ccagttaatt ttcttatttt tatctttttt tgtaagaaga tggggat 527 3 441 DNA human 3 gatcttgtca acatctgccc agcccaagac gctgaccttg ccttctctcc cttccaggat 60 gacacagaac catacttcat tggaattttt tgtttcgagg ctggaattaa aatcattgcc 120 cttgggtttg ccttccacaa aggctcctac ttgaggaatg gctggaatgt catggacttt 180 gtggtggtgc taacggggta agtggcgcgt gctatacgct ttggatttaa ctagctgaag 240 gattacgagg cttttggttg gtgtggtccg ggccaggctc aggaaggctg agcccttgtg 300 ttctccctcc ccttgttatg cgcctgcctc ctttctgcca acaccccacc tccatgtctc 360 agctgtatat tacagcagat gctttctgtt acaattaaaa taatagctca ttattgttgg 420 ctgcttccag agtgctttat g 441 4 259 DNA human 4 aaaactgagg ccagtggtgt cgagtcacct gcctgtggtc acccaaccaa tacaggacag 60 cttggaatcc caagcacccc cgccctgctg tctgaccccc aaaacccacc ctctgttctc 120 cattctggct tctttctttc agcatcttgg cgacagttgg gacggagttt gacctacgga 180 cgctgagggc agttcgagtg ctgcggccgc tcaagctggt gtctggaatc ccaagtgcgt 240 gagtttccga ccctgacaa 259 5 399 DNA human 5 cttaatattc cctcaggaac acacctgctt tgtctgggag agacctgggc gtcttggtgg 60 cggggttttg ggggtacttg ctcatgggct tatggggcct ctctctgtgt ccccccaggt 120 ttacaagtcg tcctgaagtc gatcatgaag gcgatgatcc ctttgctgca gatcggcctc 180 ctcctatttt ttgcaatcct tatttttgca atcatagggt tagaatttta tatgggaaaa 240 tttcatacca cctgctttga agaggggaca ggtaggtcca cggagcatga tgcatctttc 300 cagttttctc cttcagggac aagctcttgg gaggattagg caggggtgtg cttctttctc 360 ctggcagctg ggaggaccgt ctccttcaga gagcactac 399 6 586 DNA human 6 ttttttccct tcccttttgt agatgacatt cagggtgagt ctccggctcc atgtgggaca 60 gaagagcccg cccgcacctg ccccaatggg accaaatgtc agccctactg ggaagggccc 120 aacaacggga tcactcagtt cgacaacatc ctgtttgcag tgctgactgt tttccagtgc 180 ataaccatgg aagggtggac tgatctcctc tacaatgtaa gtgatgctgg gacagtgtgt 240 gtggacaatc agagtctcag ggaggtggcc tcctgggacc agtgagactc caaggctgca 300 atggagggac cctgagctgg gaaaggcagc ccaaggacaa cacagcccca ctgaagctgg 360 cctgaggctc aggcttttga agattacagg ggctcatgag cagaactcta actatagggc 420 atagaagtct ggagggcccc cagatgcaac atcatttttc attgtgcaag tgtttagata 480 taattttaga tttttgaata cggaaaggtt atgtgatcca aaatccaaca cagataaaag 540 atagagtaat atctttggac gtaggcgagg ggtccctgcc ctgagg 586 7 387 DNA human 7 tttcttcaga aaacggttcc ttcctccatt tccccctctg ggatgccaga gccccagaac 60 tccacaagcc aagaacattt aagacagagc cacaagagaa ccgagcttcc ccttccctca 120 cctgtcaggt tctatctgag tcccagtcaa ctctcacctg ctttccctcc tcacacccta 180 cagagcaacg atgcctcagg gaacacttgg aactggttgt acttcatccc cctcatcatc 240 atcggctcct tttttatgct gaaccttgtg ctgggtgtgc tgtcagggta agtttctgct 300 actccccacc ccatcccact cactcctctt tgctaacttc tttccaagta gaggccattg 360 aagctttgtt ttcattcact agacaga 387 8 412 DNA human 8 cccagtcttt tcccagaagt cctgactcct cctgttgaaa actcctgacc tccagggact 60 tctgaatccc caaacacaca cacacacaaa cacacacaca cacacacaca cacacacaca 120 caaacacaca cacaaacgtt tcctaacatt ttcaaaacag ccatactctg gcttttctat 180 gcttctccag ggagtttgcc aaagaaaggg aacgggtgga gaaccggcgg gcttttctga 240 agctgaggcg gcaacaacag attgaacgtg agctcaatgg gtacatggaa tggatctcaa 300 aagcaggtga ggccctttca tcctggggcc cagggatgga gatcccaggc cacagagtac 360 aaagagagtc atgcagtttg gagaaggcta agctgggagg gttatgatgg ga 412 9 611 DNA human 9 gagtaggaag ttagaggcag ggtggtcagg gaaggcttct ctaaggaagt accctctgag 60 cagagagacc tgaaggacgt gaagaaggaa gctgtgggga tgtcaaggga aggggcattc 120 caggcagaga cagcaagtgc aaaggccctg agctaggaac gtatttgaga cacagcaagg 180 aagccagtgc agctgaaaca gagtgagagg tggggacagc tggaggagag gaagacagga 240 aggtgatgga gatcagatca agcaggggct tataggctgt ggtgtggaca ttggttttta 300 ttttgcgcga ggtggggaga atgttggcta ttgctactgt tgcggaggtg gggcttgaag 360 tcacaaacca cccagcagca tgttttttgg tcggttgagc tgtcaccatc agtcagcaga 420 gaatgggggt ggccgggcag acccttcttc ctggtccaag ggagaactca tcctccaaat 480 gcaggagctt aactctgtgc tcttcctctt cagaagaggt gatcctcgcc gaggatgaaa 540 ctgacgggga gcagaggcat ccctttgatg gtaactgctc taaacccacc tcaggggtgg 600 gtcccagggg a 611 10 656 DNA human Unsure (638)..(638) n = g, a, c, t or u 10 ttaatccaag acacactgtg tgtcctatat ggtctgtgtt cgaaaaaggg taacgtcttt 60 ttctcttgcc atgtttccat tgttaggagc tctgcggaga accaccataa agaaaagcaa 120 gacagatttg ctcaaccccg aagaggctga ggatcagctg gctgatatag cctctgtggg 180 tgagtccctt cctctgccac ctatcagttg ttcatcacct atcgcccaag agacatggtg 240 gggtgggggc agagggcttg caaaccgtgc tgcctggatt tgggtctcag ctccaccctt 300 tcccacctgt gcgtgtgtcc tgggcagatt acatcattat gggaataaca tccgtgccta 360 gcttctcatt attttgtggg aattcaacta aatgatcccc atgaagcatg gcaaaccagc 420 acctggcagg gacgaagctc ccagtcaagt tggtgaatgt ttgtgactca ttcgggaagt 480 atcatggggg acctgcttat attaggtgct tggttgcaaa caaacaaggc agtcacgagg 540 ctgagctggg aggatcactt gagcctggga agtggaggct gcaataagcc attattgtgt 600 tactgcactc cagcctggca cagaaaaaaa aaaaaaanac aaactgagcc agcaca 656 11 778 DNA human 11 gatcacttct aaagttaaat gtccatggga aaacagtctc atccacatct ctttctggag 60 gccttccaag cgtgctccat gcagctctgt tgcctgcccc tgcatcaggg aatggaggct 120 ctgctttatc ctgccctgtg gtgtgactcc cagaggcatc agatgtggct gggagtggga 180 gacatggaaa attggctcct gcaacagaga actatcagcc ttcccatcaa ttggttactt 240 ctaattctgt tatttttcag gggcactgtc ttctcataag ctccatctat gcaaaactaa 300 gcccatgggt catgatggtt ccctcaggcc agaggcttgc tggagagact aatggatccc 360 ctggctaaaa tctgtgcttg ggctgcacat tggttaattt cttctgaagg aacagcctga 420 gcctgacatt ctccatcttt tccctggcag gttctccctt cgcccgagcc agcattaaaa 480 gtgccaagct ggagaactcg accttttttc acaaaaagga gaggaggatg cgtttctaca 540 tccgccgcat ggtcaaaact caggccttct actggactgt actcagtttg gtagctctca 600 acacgctgtg tgttgctatt gttcactaca accagcccga gtggctctcc gacttccttt 660 gtgagtatca cccagcccca cccctgccaa ctccctgatc cctccctcac accctttttc 720 cacttctctt tctctggtag tatgtgtatc ttctttggtc ctcattgaat ctgccctt 778 12 626 DNA human Unsure (27)..(27) n = g, a, c, t or u 12 gatcacttgt ggccaggagt tcaagancag ccagggcaac atagtgagga cccccatctc 60 cacattaaaa attttaaaaa gaaaaaagat aagtcagaag ttgggtgtgg tgacacatgc 120 ctgtagttct agcatgttgg aggccaaatc agggaaactg tttgaggcca ggagtttgaa 180 accagcctaa cagcatagca agacctcatc tctacaaaaa ataaaaagtt taaaaatgat 240 aataaaagga aagtcagagc cacctggaac ccctaccctc agcaagccta acctcctctc 300 tgtttcctcc ttctcccttc tagactatgc agaattcatt ttcttaggac tctttatgtc 360 cgaaatgttt ataaaaatgt acgggcttgg gacgcggcct tacttccact cttccttcaa 420 ctgctttgac tgtggggtaa gtgctcttgt ttctaagagt tcatttctcc agctcttgcc 480 tggaatgaca gatacctgga cacattaaag ggagaaaggt aaagtcaccc ctgaatatga 540 gagactcaga tggatgcaga aggaatgaga aaacaatcca aacactggca aggatacagt 600 gtacccagaa ccctcaacca ccgcca 626 13 976 DNA human Unsure (5)..(7) n = g, a, c, t or u 13 gatcngncat gcacaccagc ctgggtgata agagcaagac tcctctcaaa ataaatgaat 60 aaataaaaat aaataaataa ataagaggcc gggtgcagtg gctcaatgct ttggaaagtg 120 gaggccaaca gttggagaga ccaaagcagg aggatggctt cagcccagaa gtttgaggcc 180 mgcctgggca atactagcga gacactatct ctataaaaat gttttaaaat tagccagatg 240 tggtggggca cacctgtaat cccagctact caagaggctg aggtgggagg atcacttaag 300 cccaggagga cagtgctgca gtgagctatg attgcgccca ctgcactcca gcctgggtga 360 cacagtgaga cccggtctct atagataaat gaatggatga atgagggggt caaggatcct 420 cacccggctt ccatttggag ggaggagttt ggttgagttc ttgcaaggtt ggtacctagg 480 aaatgcttgc cagttctgga gcccagacac tgtccctgga catgagacca ggttctctgc 540 cctaggttat cattgggagc atcttcgagg tcatctgggc tgtcataaaa cctggcacat 600 cctttggaat cagcgtgtta cgagccctca ggttattgcg tattttcaaa ctcacaaagt 660 aagtctttgg ggttcctgga catttgtaca gggggtgggg atgggggaca tggtggggcc 720 gcctccagaa agttgggaaa gtgagcctcg tgtttcgagg gctgactccg gggcctgcct 780 wccccgcctg gcctgagtcc tcgcctggsc tctgtcggca ggtactgggc atctctcaga 840 aacctggtcg tctctctcct caactccatg aagtccatca tcagcctgtt gtttctcctt 900 ttcctgttca ttgtcgtctt cgcccttttg ggaatgcaac tcttcggcgg ccagtaagtc 960 cttcacagga attcaa 976 14 1110 DNA human Unsure (620)..(620) n = g, a, c, t or u 14 ccctccacgt gcaggctgcc ttcctcgtag cccagacacc catttgcggt cacccaaatg 60 ggcagggccc tgggtaccac tcagggtttc ctggggacag agatgatgga aacgttcgtt 120 tccttggaga tgagatactg agccacaccc tcagagcacc ccgggtgggg ccaacgtgaa 180 atgtctgtgt cctccctgca ggtttaattt cgatgaaggg actcctccca ccaacttcga 240 tacttttcca gcagcaataa tgacggtgtt tcaggtacag cctccacctg gccccacggg 300 ccaacacctc tcagtgtcac agatgaaagt gcctgctcca catccaaggg gcttccctga 360 actcctcctt ctctacctgg ccttttcaca ccactttgaa acacagattt tatggttatc 420 attattcaat tatggtgagg ccaacagatc aggagatgaa tgtcattgga aagatagttt 480 gtggctgggc acggtggctc acacccataa tcccagcact ttggccaggt acggtggctc 540 acacctgtaa tcccaacgct ttgggaagcc caggtgggcg gatcacttga gatcaggaat 600 tcgagaccag cctggccaan atggtgaaac cccatctcta ctaaaaatac aaaaattagc 660 cgggcgtggt agcacatgcc tgtaatccca gctactcggg agatgaggca caagaattgc 720 ttgaacctgg gaggcagagg ttgcagtgag ccaagatcgc gccactgcac tcmagcctgg 780 gcaacagagt gagactccat ctcaaaaaag caaaagaaaa aaaaaaccac tttgggaggt 840 caagatggga ggactacttg aggccaggag tttgagacaa gtctgggcaa catagtgaga 900 ctccgtctct gcaaaaaaat wataataata attagctggg catggtgata catacctcct 960 agctactagg gcagctgaag tggaaggatt gcttaagccc aggaggttga ggctgcagta 1020 agctacaatc acaccactat actccagcct gggcgagaga gcaaagccct gtctcaaaaa 1080 cgaaaagaaa gtttgttata ctcacagatc 1110 15 982 DNA human 15 gatcctccca ccttggcctc ccaaagtgct gggattacag gcatgagcca tggcatgcgg 60 tctcttcctg ttcttataag ggcactaata ccatcatgaa gtcccccatg acctcatcta 120 accctagtta cctcttaaag gccccatctc caaataccat cccatcatag gttagggctt 180 caactcatga atttggaggc gggcacaatt tagtccataa caaatcccct taatcacatc 240 aagtaagaca gagttacagg agggtctgtg actcctccag ggtcccattt tcctagaagc 300 caggctaaga gccccacgac gcaggaacgg ccctttctac tcgcaaacaa agagaaaagc 360 caaggagaag ccaacacgga gtctggctct gcaaaccggg caggattgtt aaagacctcc 420 tgggctcggg gatggggtgg gcggattccg gctccacagc tgcatctcca aggggcccgt 480 ggctgagagg ggggttggct gtgtgtttct tcctcccctt tcagatcctg acgggcgaag 540 actggaacga ggtcatgtac gacgggatca agtctcaggg gggcgtgcag ggcggcatgg 600 tgttctccat ctatttcatt gtactgacgc tctttgggaa ctgtatcctt catggagaga 660 gagaagggga caggcctgga cctctggcag aggagaggtt gcaggggctc aagggagggt 720 actgagagac ccagataccc agggcccaag tggtgtccca ccagtggttg cttttcctga 780 ctcagacatt tgcagacacc ctcctgaatg tgttcttggc catcgctgtg gacaatctgg 840 ccaacgccca ggagctcacc aaggtggagg cggtgggaga atgtttctct ggcaaagtta 900 ccacctgccc atggcagatc aagcactttt ttggattaac tgagccacag gaaataacat 960 tttcaaatag atkaaaaaga tc 982 16 314 DNA human 16 ccttggttct gattggtcga aatatttcaa atgttgcccc tggtcagcaa cagggtcaga 60 agtgagtccc caaggcctag ttcatgtttt gtgaacaaag attccacgtg ccttttcagg 120 acgagcaaga ggaagaagaa gcagcgaacc agaaacttgc cctacagaaa gccaaggagg 180 tggcagaagt gagtcctctg tccgcggcca acatgtctat agctgtgtaa gtcccctaat 240 ccctgggatg ctaccctggc tcctgaacgt gtccgaccac tatccaggca cagatttggg 300 aagcagtggg ggtg 314 17 1113 DNA human 17 gcccctagcc aggtgggagc catggagggt tcttgagcag aggaggctgg gacctgactc 60 agatgctcac agactcctag cattcaggtg gggagtagag ggtggagagc aggagtggga 120 ggctgagatg tgggttggtt cgcctgggtc atccatccaa gctacagtgc ctagcaatgc 180 tctaagctcc tgtgaccatg ccactgcagg aaagagcaac agaagaatca aaagccagcc 240 aagtccgtgt gggagcagcg gaccagtgag atgcgaaagc agaacttgct ggccagccgg 300 gaggccctgt ataacgaaat ggacccggac gagcgctgga aggctgccta cacgcggcac 360 ctgcggccag acatgaagac gcacttggac cggccgctgg tggtggaccc gcaggagaac 420 cgcaacaaca acaccaacaa gagccgggcg gccgagccca ccgtggacca gcgcctcggc 480 cagcagcgcg ccgaggactt cctcaggaaa caggcccgct accacgatcg ggcccgggac 540 cccagcggct cggcgggcct ggacgcacgg aggccctggg cgggaagcca ggaggccgag 600 ctgagccggg aggaccccta cggccgcgag tcggaccacc acgcccggga gggcagcctg 660 gagcaacccg ggttctggga gggcgaggcc gagcgaggca aggccgggga cccccaccgg 720 aggcacgtgc accggcaggg gggcagcagg gagagccgca gcgggtcccc gcgcacgggc 780 gcggacgggg agcatcgacg tcatcgcgcg caccgcaggc ccggggagga gggtccggag 840 gacaaggcgg agcggagggc gcggcaccgc gagggcagcc ggccggcccg gggcggcgag 900 ggcgagggcg agggtcccga cgggggcgag cgcaggagaa ggcaccggca tggcgctcca 960 gccacgtacg agggggacgc gcggagggag gacaaggagc ggaggcatcg gaggaggaag 1020 taagtggagg tgacctcgaa tccgcagaat gacggtaaca ttaataatac aacagccaaa 1080 gtagcacgtg ctgtgtattt gttataaaat ata 1113 18 590 DNA human 18 gtcctgaaac tttgcctttt aatcctaaat cattgttggt tctttttcat tcacttgcct 60 tcctcagaga gaaccagggc tccggggtcc ctgtgtcggg ccccaacctg tcaaccaccc 120 ggccaatcca gcaggacctg ggccgccaag acccacccct ggcagaggat attgacaaca 180 tgaagaacaa caagctggcc accgcggagt cggccgctcc ccacggcagc cttggccacg 240 ccggcctgcc ccagagccca gccaagatgg gaaacagcac cgaccccggc cccatgctgg 300 ccatccctgc catggccacc aacccccaga acgccgccag ccgccggacg cccaacaacc 360 cggggaaccc atccaatccc ggccccccca agacccccga gaatagcctt atcgtcacca 420 accccagcgg cacccagacc aattcagcta agactgccag gaaacccgac cacaccacag 480 tggacatccc cccagcctgc ccaccccccc tcaaccacac cgtcgtacaa ggtgagaccc 540 tctgctctca catcactggg caggggacct ggcgtcctgg agccagaggt 590 19 340 DNA human Unsure (1)..(340) n = g, a, c, t or u 19 ggagtacacc gaggagttcc cagagacttg tgggaaattg tggagggagc cctgtgttgg 60 ttcttgtccc aacagtgaac aaaaacgcca acccagaccc actgccaaaa aaagaggaag 120 agaagaagga ggaggaggaa gaagacgacc gtggggaaga cggccctaag ccaatgcctc 180 cctatagctc catgttcatc ctgtccacga ccaacccgtg agtatggccc ccgagcagag 240 ggcagggggg gctgggtctc ccaccagggt ggcggaannn nnnnnnnnnn nnnnnnnctc 300 ccaccagggt ggcggaagtc aggccagatt agagggcaat 340 20 477 DNA human 20 gatctcagta gtggtaggta acatgagatt atggaagaaa agggtttgtg agcctgtggt 60 ctgagtggac ctctgcacgc ccatctgtct ccaacagcct tcgccgcctg tgccattaca 120 tcctgaacct gcgctacttt gagatgtgca tcctcatggt cattgccatg agcagcatcg 180 ccctggccgc cgaggaccct gtgcagccca acgcacctcg gaacaacgtg agtcccacag 240 agcacacccc ttcctagcct ggctgctctg cctcaggcca ctttctcctg catccaaaat 300 gctcataggt agggtgggat gttggggtca cccctaggca tagcccttat ggctgctggt 360 tgagagggga agctctgatt ccttggggat gctcttggga gcaagacatt ccttgaggca 420 gtttctctgt gagcctggtg gggtggaggt ggcccagagt gactggggct gaaaatt 477 21 168 DNA human 21 gatccactgc tctcttgctt ttatccctta caggtgctgc gatactttga ctacgttttt 60 acaggcgtct ttacctttga gatggtgatc aaggtgagtg cagattataa gtgagaacac 120 acggtaattt ttttttttaa gcaagtgcag ggctgggcac agtggatc 168 22 368 DNA human 22 gatctaagag ccggcaagcc agagctggct tccatcaggc aaaggggggc cgcctcatgg 60 ggcaggggct ccccactcct ccctgggagt cctctggcca ctgcccatcc ctgcaagatg 120 aggtggcctc attggcttcc ctgcctctcc ccgagaggct agagagtggg tggcagcacc 180 ccagggtggg gatcaggtgg gggttctgag caccctctct tctcccccac agatgattga 240 cctggggctc gtcctgcatc agggtgccta cttccgtgac ctctggaata ttctcgactt 300 catagtggtc agtggggccc tggtagcctt tgccttcacg taagtctctt cgcaagggtt 360 tcctcttg 368 23 515 DNA human 23 gatcttaacc ccaagacact tcatctaaag gaaaaactgc cataatacac agattatttt 60 aggtcagctc actttactgc catctgctgg gaagttgtaa taatacaaat atccatacac 120 gatggctagg atgttatcag cacctccttt aatgtgttgt ccttgagcag tgtacaacct 180 gctcagctgt acatgataac cctgacagtc ccccccaccg caccccacca tctcccaatc 240 tcaccttgag ctttggcagc cgcttgatgg ttttaagagg tcgtagcacc cggaggactc 300 ggagggattt aatcgtgttg atgtcttttc ctttgctatt gccactgtgg aggaatgttt 360 aggtgggaag aagggaagag aggaagcaga ggtcaggttg ggtagggggc agcccacagc 420 tccatgggac cctacccttc ccaggcctag aagtctgggg tgagcttggc acaagcctgc 480 cctttcctgg tgaagagtgg tccattttac cctgt 515 24 406 DNA human 24 ggccactgga ggcagaaggt tggcaggtcc ccagcccctc atgctctctg tcaactccac 60 cccacaggct gtgtttgact gtgtggtgaa ctcacttaaa aacgtcttca acatcctcat 120 cgtctacatg ctattcatgt tcatcttcgc cgtggtggct gtgcagctct tcaaggggaa 180 attcttccac tgcactgacg agtccaaaga gtttgagaaa gattgtcggt gggtctccgc 240 tttccagcac attcccattg gaaccagcag gtgggcaggg gggaagtggc tagaggcatt 300 ggccacttgg gctcagagac tggagaagtg atgagccttg gaagtgactc agttgcaacc 360 agcttggatc aagggtagaa agaaaaccgg ttttagaatt tgagtc 406 25 516 DNA human Unsure (421)..(516) n = g, a, c, t or u 25 gatctcaaac tcctggcctc aagtgataca tctgccttgg cctcctaaag tgttgggatt 60 acaggcgtga gcaccatgcc cggcctccaa gacctttctt attgctaagc tctcaggccc 120 tttatcctcc tgctccccag ggctcctcct ggatagattt ccagtcgggc cacttactgt 180 ggccagcctt ctcccgtgga cacggtgaag agggtcagca gagcccacag cacattgtcg 240 taatggaatt catacttctt ccactcccgg tctcgcgcct tcacctcatt cttctcgtag 300 aggaggtatt tgcctctgcc acagagagtg gggactgtta gtaaatggga aagaggggct 360 gtcttgcact tgtctttggt tatcagagac agggggaggg aaaggaagag tggtccacca 420 ncctagactg cttgggaagc agtgacttcc catcctgcca ccatgtgttc ctgtgcttca 480 taggggatgn cgtgtgcaat ctacttttna ggataa 516 26 489 DNA human 26 accttcctca tcacccttgg gtccctgtct ctctccttcc tgccccttcc ctctccctgc 60 cccattcctt gcagggtcct caagcattcg gtggacgcca cctttgagaa ccagggcccc 120 agccccgggt accgcatgga gatgtccatt ttctacgtcg tctactttgt ggtgttcccc 180 ttcttctttg tcaatatctt tgtggccttg atcatcatca ccttccagga gcaaggggac 240 aagatgatgg aggaatacag cctggagaaa aatgaggtgc cacttccaat tccatctgtc 300 ctttaaaaac tggggacaca cacaaacttt aaaacacaca caacacccag gaaccccttt 360 ctaggggtac ctgggggagg gaacagaagc attgtcccaa ccgaatccag tcttcagggc 420 agcccttcat ggagtttcag aggaaacaca tcatatagtg tatgtatcag tcagtttaga 480 ctaggttat 489 27 512 DNA human Unsure (1)..(512) n = g, a, c, t or u 27 tagcccatgc aanaatgggg aaatgncagt gcaagttttg gcagttgntg acatctcaag 60 caactgtanc tgttgggata agaaagcaat ggtgagaagg aanagaganc ccaggaatcc 120 tggctggggg caananaggc agagactcaa gcagaagcac ttgagaaccg cgacgagtta 180 gacagagggt gcccggtgta cagccacctt cctcctgcct ctgccgctct caccactggc 240 ctctctcccg cagagggcct gcattgattt cgccatcagt gccaagccgc tgacccgaca 300 catgccgcag aacaagcaga gcttccagta ccgcatgtgg cagttcgtgg tgtctccgcc 360 tttcgagtac acgatcatgg ccatgatcgc cctcaacacc atcgtgctta tgatgaaggt 420 aagtgcccca caccagcccc cagcactant taacccccac ctcgttcctg cctctaccct 480 gataaaatga aaccatttgc agatttccca ga 512 28 411 DNA human Unsure (306)..(309) n = g, a, c, t or u 28 gggtctttcc tgaactgtgc ctcctaccag tgaggttgct cagaccttgc ctggggctgg 60 agtgttgcct ggagaacagc catgaagctg acctccccac ttcccacttc ccacccctgc 120 tcgctgaccc ctgctactcc tgcttctttc ccctagttct atggggcttc tgtggcttat 180 gaaaatgccc tgcgggtgtt caacatcgcc ttcacctccc tcttctctct ggaatgtgtg 240 ctgaaagcca tggcttttgg gattctggta agtaccacct tggggctaca gctatgggct 300 tggaanaanc ccaaggggga acaatgggtc ctggatgatg gtctcccaac gtggccccaa 360 gaaccccaac ctcaagggtg gcttcagtat cctgcccagt ggccacagat c 411 29 420 DNA human 29 ctgtcccggg cactccgctg atgggcaact gtgcctctaa catgcaccgg ccagcctagg 60 gggccgggaa ccaagccctc tgttggcatc tctgtcttgt gggtccccat tctagaatta 120 tttccgcgat gcctggaaca tcttcgactt tgtgactgtt ctgggcagca tcaccgatat 180 cctcgtgact gagtttgggg taagtctccc tccagcttct ctctgggtga ctctgggctg 240 gacgaggcag gcggcagggg gcgggggagc ggtcccagag gcagtgtgtc ccggaagcca 300 tagctgcttg agccagcact tggccatgac cagagaggga gaactggggc cccggggaca 360 agggcagccc ctcaggaggg cattgtgggg agatgggggt aacaaagctt ggctgtaggg 420 30 342 DNA human 30 ttaatagtgc tttctctctc cctccttatt tggggtctgg cttgcttttt tcctgttggt 60 tggcttcatg taggggcctc tgtgagtggt gacagctctg agcctttggg gtgggtggat 120 ggtcacccct cttcctccat ctccccagaa taacttcatc aacctgagct ttctccgcct 180 cttccgagct gcccggctca tcaaacttct ccgtcagggt tacaccatcc gcattcttct 240 ctggaccttt gtgcagtcct tcaaggtgag tcctcgtccc tgctgctggc ccaggggctg 300 agaagacagg tgaccctcat gctctggctg aatgtagaag tc 342 31 559 DNA human Unsure (536)..(536) n = g, a, c, t or u 31 cccccaagaa gaatgcccac caagccctgg aaggactctg gcacgtggca tatggccacc 60 caacccagtg gggcagagca ctgggacaag ggaggaagac tgcagtgcgg ctgagggacc 120 cccagcactc ttcttcattg ccttttttcc caccaggccc tgccttatgt ctgtctgctg 180 atcgccatgc tcttcttcat ctatgccatc attgggatgc aggtgagtgt cgtgtcccta 240 aggttcccag agcctcccaa ggagggcagc cacccttaga aaggggtggg tcagaggagc 300 ctggttcaca gaagcagcca tggaggttga gctgggtttc ccagaagcca ctggaggaat 360 ggcagcccct ggtcgtcacc cwmcaattcc acaggtgttt ggtaacattg gcatcgacgt 420 ggaggacgag gacagtgatg aagatgagtt ccaaatcact gagcacaata acttccggac 480 cttcttccag gccctcatgc tctcttccgg tcagaagggg acctgctctg ataatnctgt 540 ttccgtgggg tggggtgcc 559 32 316 DNA human 32 tcagagccat gctcactgtg tgctccactc ctgaggaggc gttggtacca gtcagggctg 60 ggtgtccgag tctctgattt ctccctgtcc tcaggagtgc caccggggaa gcttggcaca 120 acatcatgct ttcctgcctc agcgggaaac cgtgtgataa gaactctggc atcctgactc 180 gagagtgtgg caatgaattt gcttattttt actttgtttc cttcatcttc ctctgctcgt 240 ttctggtgag tctgtggaca ctgtgagggc cgtctgggct ccctaagcct ggcttccttt 300 cagggagtgg ttctgt 316 33 694 DNA human Unsure (413)..(413) n = g, a, c, t or u 33 gtgtagtgag aactcacctc tccattcccc agtctctttc tgtctctgtc tcatttcctt 60 tccccatctt ctctctatcc ctctctccat ctggggcctc tgtgtctgtc tttgggtctg 120 tctgtccgtc tgactgtctg tatccttctc acttcactca ttcattccct cggtctctgc 180 cccattctct cttggtcccg ggtccccaca gatgctgaat ctctttgtcg ccgtcatcat 240 ggacaacttt gagtacctca cccgagactc ctccatcctg ggcccccacc acctggatga 300 gtacgtgcgt gtctgggccg agtatgaccc cgcagcttgg taagaagtca ccccgaatcc 360 tccagccaca atactcacct ctccctggaa ctggaacacg ggctaggcta ggnccccaga 420 ctctggagca ctgaactcct ggggctccta gcaggggtct cacaggttca gtcaggagag 480 aagatataag aatcatcacc cttgcatacc ccagattaaa cacgtagggt gccaacctgc 540 ccaaaccctg gaggactttc tgggaaatga ggagggcgtc aaccatgaga tgtctgaaga 600 gccctctcct cctacgagtc tctcctgtct ctcactgtga agtctccaga tggtgaggat 660 cgattagcca ggctccagga gaaaccaaca gact 694 34 474 DNA human 34 aagggaggtg cctgcagtcc cgaactcgac tgacatccta cacccctggg tctccccagt 60 gtctgggaat gtactgggaa ttcacttgtc cccagtctct cccactcctt gaagccaggg 120 acaccccagc ctcgggcatc atgacctcgt tgtgtgccca gggagcccgt gtgaacccat 180 tgcctgcact aacccccttt cttctccttt cagcggtcgg attcattata aggatatgta 240 cagtttatta cgagtaatat ctccccctct cggcttaggc aagaaatgtc ctcatagggt 300 tgcttgcaag gtttgacttc cactaaaacc tgctagcatc catggaatga gtgtggcttg 360 gggttcttca atatatatat ttcatatata tatatatata tatctctctc tctctaaaaa 420 aacagagcca tctctctttc ttgcattaaa ctagaaaact ctcttagcca acag 474 35 413 DNA human Unsure (323)..(413) n = g, a, c, t or u 35 cctgggtagg ggcgggcgcg gctcacggga gacccaggag ggatgcctgg gaatgactgc 60 gcttgccttg ggttttctgt agcggcttct gcggatggac ctgcccgtcg cagatgacaa 120 caccgtccac ttcaattcca ccctcatggc tctgatccgc acagccctgg acatcaagat 180 tgccaagggt aaggaaggga caggggcggg cacagacagg cgtgacaggg tggaactggg 240 gatctcctcc ctaccccaaa ctagaggatc tgctgtcacc acccggatct tcattcactc 300 ttccattcat tcgttccaca ggnntttttg gnnnttggnn ntttggtgtt tttttttttt 360 ttttgagaca gagtcttgct ctgttgccca ggcagcagtg cggtgacatg atc 413 36 636 DNA human Unsure (332)..(332) n = g, a, c, t or u 36 gggtctcgtt ctcgggagcc tatggctttg cagctgaccc agagtccagc tgacacccag 60 gcaggcagtc agggtctgtc tacaccccca ttgcaggagg agccgacaaa cagcagatgg 120 acgctgagct gcggaaggag atgatggcga tttggcccaa tctgtcccag aagacgctag 180 acctgctggt cacacctcac aagtgtaaga gctgagccca gccctgggat ccaatccacc 240 aggacagatg gagggggagg gaaaggggag gcctggggag agtgttggct gggctggtat 300 acacagggac ccaggacaag gtccccaaag angcctgccc ttggtgagct caccgtgtgt 360 gtcccccagc cacggacctc accgtgggga agatctacgc agccatgatg atcatggagt 420 actaccggca gagcaaggcc aagaagctgc aggccatgcg cgaggagcag gtgcgctgtt 480 cgccgctctg gggacatctg ggctggggac agtggcttgc atgtcaccac gggaaccaac 540 tggaatatga gggtggctga gccccagggc aggtccctga aaagtagggg ctggtgcaca 600 gcagctcaca cctgcaatct cagtgctttg agaggc 636 37 829 DNA human 37 gatcttcagg gccatgggag ctgcaggaag gactctggct ttttccccaa gcaagtggga 60 gccatggagg gttctaagca aaggagggat aggacctgac tcaagtgctc atgggcgccc 120 tctggtggct cttgtggaac agtggggttg aaggtaggag cgggagacct gggagaaggt 180 gcctgcagtg agagatgagg acgcgggacc aggctggggc tatgacttgg gtggaggagt 240 gagaagtggt ccagttctgc gtggaattgg aagggtctag atggatgaga cctgagagag 300 tgtgtgtgtg tgtgtgtgtg tatactgggg atgtcgcaat gccttctggg taccaccgtc 360 caccacccca cccttgtcca cacactgctc tctgccccat tccccaggac cggacacccc 420 tcatgttcca gcgcatggag cccccgtccc caacgcagga agggggacct ggccagaacg 480 ccctcccctc cacccagctg gacccaggag gagccctgtg agtgtcaccc ctgccaggga 540 ggtggagtgt gggggtgccg tggtccccac gttctggaag ctgcccaagc gcccactgct 600 accccggcct ctgtccccca tgcaggatgg ctcacgaaag cggcctcaag gagagcccgt 660 cctgggtgac ccagcgtgcc caggagatgt tccagaagac gggcacatgg agtccggaac 720 aaggcccccc taccgacatg cccaacagcc agcctaactc tcaggtgcct ctgtccccca 780 actccccaat ggctcccagg gcccgggtgg ttgcggtgga aggaaccat 829 38 801 DNA human Unsure (161)..(161) n = g, a, c, t or u 38 tcactgcaac ctccaccttc cagtctcaag tgattcctcc tgcctcagcc tcccaagtca 60 ctggattaca ggcgcccacc accatgctca ggtatttttt tttgtatttt tagtagagac 120 ggggtttcac aatgttggtc aggctggtct cgaactgctg nccattgtga tctggaggtc 180 aggccccaga gctcatctgg ctttgccatt cgtccgcagt ccgtggagat gcgagagatg 240 ggcagagatg gctactccga cagcgagcac tacctcccca tggaaggcca gggccgggct 300 gcctccatgc cccgcctccc tgcagagaac caggtgaggg ctttcaccac tgccctgggg 360 ctggacccct cactctgcac tgggtagggc caggcccccc cacaagcagc ccagtgcatc 420 ccctcctgcc ggactcaggc ctgggtaggg actccttcag tctctgaagc agtctgcagg 480 ccccacccac cacctggtca cacctggagc acctgcagac cctcctccct cacagaggac 540 agagaggaaa gtgctccccc tggggcagag ggcagtggcc actgcaaaat ggtctctggc 600 tgccctggtt ggaggctgca gacaggggag gttgtggaar atttgtgggt gcagcagggt 660 tcaacagggc cagctgagac ctgccacgaa gawcctttga ggccaggagt ttgagaccag 720 gttgggcaac atagcaaaac cctgtctctt tttttttttt gagacggagt ttcactcttg 780 ttgccccagg ctggagtgac a 801 39 329 DNA human Unsure (177)..(177) n = g, a, c, t or u 39 cctcctcact cttccctctt gcctttatat ttattttctt ctttctgttt tttctgtgtg 60 caccatccat ggggctgtga cagaggagaa ggggccggcc acgtgggaat aacctcagtg 120 tatgtacggc ctgcccaggg cccagcaggc tccggccccc tcttcctccc caccccncct 180 ccagggagtc ccgtaatctc taccggtccc cggaccccac cctttctttg gcaatcgcac 240 cctctcccct ccatggagcc caatccttgt gtgtggtgtc ctgtgtgtgc cctgacccat 300 aagcctggtg gggcggccat ccccatcct 329 40 554 DNA human 40 gatcaggggg agccaaggcc ccatggcatc ccctggcccc tgccccagga tggtcacacc 60 gcagtcaccg aaggccacca ccaggctgcc acaatggggc aggaaggacc gggaccactt 120 ggtgctagct gctgacccca gcccaccggc ctgtcccctc ccccagacca tctcagacac 180 cagccccatg aagcgttcag cctccgtgct gggccccaag gcccgacgcc tggacgatta 240 ctcgctggag cgggtcccgc ccgaggagaa ccagcggcac caccagcggc gccgcgaccg 300 cagccaccgc gcctctgagc gctccctggg ccgctacacc gatgtggaca caggtgggca 360 gccctgtggt gctcagggac aagcagaaca gaggagagga gaggggagga gaaggcaggg 420 cggaggagac actaaggaag aagaaaggga gaggcctcca tggagagggg acagagcggg 480 ccaggcagcg gctgcaggaa cctgggtact accccctccc cccaacccac tgacctgcct 540 cggttcaggg gatc 554 41 461 DNA human 41 ctgtgtgctg tctgaccctc acccggccca ggcttgggga cagacctgag catgaccacc 60 caatccgggg acctgccgtc gaaggagcgg gaccaggagc ggggccggcc caaggatcgg 120 aagcatcgac agcaccacca ccaccaccac caccaccacc atcccccgcc ccccgacaag 180 gaccgctatg cccaggaacg gccggaccac ggccgggcac gggctcggga ccagcgctgg 240 tcccgctcgc ccagcgaggg ccgagagcac atggcgcacc ggcaggtggg tgcggctgca 300 agtgacccca ggctgggctc ggccgggagg cggggaggag agaaggggat accccatcca 360 acagccactc taggcaaagg tccccggatc ccggctgtga ccacctccca tcctgccccc 420 aagccaccgg ggtgcccggc ggccggagcg gagcacggat c 461 42 664 DNA HUMAN 42 tttctcattt ctcttttcac ttttgttgtg ttggtttccg actcctcccc tccctgtctc 60 actccccctc ctcccctccc tcctccctgt ggctgttgct tttttccatt caatgtcctg 120 tgtcccccct ctcctcctcc tcctcctcct ccccctcctc cctctcctcc cggcccctct 180 cccttcgctc ccctcatctt cctcccaatc ccgtgtctcc tttgattttg ttgtatcttt 240 ttttttgatt tcctttgttt caattttcgt gtagggcagt agttccgtaa gtggaagccc 300 agccccctca acatctggta ccagcactcc gcggcggggc cgccgccagc tcccccagac 360 cccctccacc ccccggccac acgtgtccta ttcccctgtg atccgtaagg ccggcggctc 420 ggggcccccg cagcagcagc agcagcagca gcagcagcag caggcggtgg ccaggccggc 480 cgggcggcca ccagcggccc tcggaggtac ccaggcccca cggccgagcc tctggccgga 540 gatcggcgcc cacggggggc cacagcagcg gccgcacgcc caggatggag aggcgggtcc 600 aggcccggcc cggagcgagt ctccagggcc tggtcgacac ggcggggccc ggctggcggc 660 agtc 664 43 6789 DNA HUMAN 43 atggcccgct tcggagacga gatgccggcc cgctacgggg gaggaggctc cggggcagcc 60 gccggggtgg tcgtgggcag cggaggcggg cgaggagccg ggggcagccg gcagggcggg 120 cagcccgggg cgcaaaggat gtacaagcag tcaatggcgc agagagcgcg gaccatggca 180 ctctacaacc ccatccccgt ccgacagaac tgcctcacgg ttaaccggtc tctcttcctc 240 ttcagcgaag acaacgtggt gagaaaatac gccaaaaaga tcaccgaatg gcctcccttt 300 gaatatatga ttttagccac catcatagcg aattgcatcg tcctcgcact ggagcagcat 360 ctgcctgatg atgacaagac cccgatgtct gaacggctgg atgacacaga accatacttc 420 attggaattt tttgtttcga ggctggaatt aaaatcattg cccttgggtt tgccttccac 480 aaaggctcct acttgaggaa tggctggaat gtcatggact ttgtggtggt gctaacgggc 540 atcttggcga cagttgggac ggagtttgac ctacggacgc tgagggcagt tcgagtgctg 600 cggccgctca agctggtgtc tggaatccca agtttacaag tcgtcctgaa gtcgatcatg 660 aaggcgatga tccctttgct gcagatcggc ctcctcctat tttttgcaat ccttattttt 720 gcaatcatag ggttagaatt ttatatggga aaatttcata ccacctgctt tgaagagggg 780 acagatgaca ttcagggtga gtctccggct ccatgtggga cagaagagcc cgcccgcacc 840 tgccccaatg ggaccaaatg tcagccctac tgggaagggc ccaacaacgg gatcactcag 900 ttcgacaaca tcctgtttgc agtgctgact gttttccagt gcataaccat ggaagggtgg 960 actgatctcc tctacaatag caacgatgcc tcagggaaca cttggaactg gttgtacttc 1020 atccccctca tcatcatcgg ctcctttttt atgctgaacc ttgtgctggg tgtgctgtca 1080 ggggagtttg ccaaagaaag ggaacgggtg gagaaccggc gggcttttct gaagctgagg 1140 cggcaacaac agattgaacg tgagctcaat gggtacatgg aatggatctc aaaagcagaa 1200 gaggtgatcc tcgccgagga tgaaactgac ggggagcaga ggcatccctt tgatggagct 1260 ctgcggagaa ccaccataaa gaaaagcaag acagatttgc tcaaccccga agaggctgag 1320 gatcagctgg ctgatatagc ctctgtgggt tctcccttcg cccgagccag cattaaaagt 1380 gccaagctgg agaactcgac cttttttcac aaaaaggaga ggaggatgcg tttctacatc 1440 cgccgcatgg tcaaaactca ggccttctac tggactgtac tcagtttggt agctctcaac 1500 acgctgtgtg ttgctattgt tcactacaac cagcccgagt ggctctccga cttcctttac 1560 tatgcagaat tcattttctt aggactcttt atgtccgaaa tgtttataaa aatgtacggg 1620 cttgggacgc ggccttactt ccactcttcc ttcaactgct ttgactgtgg ggttatcatt 1680 gggagcatct tcgaggtcat ctgggctgtc ataaaacctg gcacatcctt tggaatcagc 1740 gtgttacgag ccctcaggtt attgcgtatt ttcaaagtca caaagtactg ggcatctctc 1800 agaaacctgg tcgtctctct cctcaactcc atgaagtcca tcatcagcct gttgtttctc 1860 cttttcctgt tcattgtcgt cttcgccctt ttgggaatgc aactcttcgg cggccagttt 1920 aatttcgatg aagggactcc tcccaccaac ttcgatactt ttccagcagc aataatgacg 1980 gtgtttcaga tcctgacggg cgaagactgg aacgaggtca tgtacgacgg gatcaagtct 2040 caggggggcg tgcagggcgg catggtgttc tccatctatt tcattgtact gacgctcttt 2100 gggaactaca ccctcctgaa tgtgttcttg gccatcgctg tggacaatct ggccaacgcc 2160 caggagctca ccaaggacga gcaagaggaa gaagaagcag cgaaccagaa acttgcccta 2220 cagaaagcca aggaggtggc agaagtgagt cctctgtccg cggccaacat gtctatagct 2280 gtgaaagagc aacagaagaa tcaaaagcca gccaagtccg tgtgggagca gcggaccagt 2340 gagatgcgaa agcagaactt gctggccagc cgggaggccc tgtataacga aatggacccg 2400 gacgagcgct ggaaggctgc ctacacgcgg cacctgcggc cagacatgaa gacgcacttg 2460 gaccggccgc tggtggtgga cccgcaggag aaccgcaaca acaacaccaa caagagccgg 2520 gcggccgagc ccaccgtgga ccagcgcctc ggccagcagc gcgccgagga cttcctcagg 2580 aaacaggccc gctaccacga tcgggcccgg gaccccagcg gctcggcggg cctggacgca 2640 cggaggccct gggcgggaag ccaggaggcc gagctgagcc gggaggaccc ctacggccgc 2700 gagtcggacc accacgcccg ggagggcagc ctggagcaac ccgggttctg ggagggcgag 2760 gccgagcgag gcaaggccgg ggacccccac cggaggcacg tgcaccggca ggggggcagc 2820 agggagagcc gcagcgggtc cccgcgcacg ggcgcggacg gggagcatcg acgtcatcgc 2880 gcgcaccgca ggcccgggga ggagggtccg gaggacaagg cggagcggag ggcgcggcac 2940 cgcgagggca gccggccggc ccggggcggc gagggcgagg gcgagggtcc cgacgggggc 3000 gagcgcagga gaaggcaccg gcatggcgct ccagccacgt acgaggggga cgcgcggagg 3060 gaggacaagg agcggaggca tcggaggagg aaagagaacc agggctccgg ggtccctgtg 3120 tcgggcccca acctgtcaac cacccggcca atccagcagg acctgggccg ccaagaccca 3180 cccctggcag aggatattga caacatgaag aacaacaagc tggccaccgc ggagtcggcc 3240 gctccccacg gcagccttgg ccacgccggc ctgccccaga gcccagccaa gatgggaaac 3300 agcaccgacc ccggccccat gctggccatc cctgccatgg ccaccaaccc ccagaacgcc 3360 gccagccgcc ggacgcccaa caacccgggg aacccatcca atcccggccc ccccaagacc 3420 cccgagaata gccttatcgt caccaacccc agcggcaccc agaccaattc agctaagact 3480 gccaggaaac ccgaccacac cacagtggac atccccccag cctgcccacc ccccctcaac 3540 cacaccgtcg tacaagtgaa caaaaacgcc aacccagacc cactgccaaa aaaagaggaa 3600 gagaagaagg aggaggagga agaagacgac cgtggggaag acggccctaa gccaatgcct 3660 ccctatagct ccatgttcat cctgtccacg accaaccccc ttcgccgcct gtgccattac 3720 atcctgaacc tgcgctactt tgagatgtgc atcctcatgg tcattgccat gagcagcatc 3780 gccctggccg ccgaggaccc tgtgcagccc aacgcacctc ggaacaacgt gctgcgatac 3840 tttgactacg tttttacagg cgtctttacc tttgagatgg tgatcaagat gattgacctg 3900 gggctcgtcc tgcatcaggg tgcctacttc cgtgacctct ggaatattct cgacttcata 3960 gtggtcagtg gggccctggt agcctttgcc ttcactggca atagcaaagg aaaagacatc 4020 aacacgatta aatccctccg agtcctccgg gtgctacgac ctcttaaaac catcaagcgg 4080 ctgccaaagc tcaaggctgt gtttgactgt gtggtgaact cacttaaaaa cgtcttcaac 4140 atcctcatcg tctacatgct attcatgttc atcttcgccg tggtggctgt gcagctcttc 4200 aaggggaaat tcttccactg cactgacgag tccaaagagt ttgagaaaga ttgtcgaggc 4260 aaatacctcc tctacgagaa gaatgaggtg aaggcgcgag accgggagtg gaagaagtat 4320 gaattccatt acgacaatgt gctgtgggct ctgctgaccc tcttcaccgt gtccacggca 4380 gaaggctggc cacaggtcct caagcattcg gtggacgcca cctttgagaa ccagggcccc 4440 agccccgggt accgcatgga gatgtccatt ttctacgtcg tctactttgt ggtgttcccc 4500 ttcttctttg tcaatatctt tgtggccttg atcatcatca ccttccagga gcaaggggac 4560 aagatgatgg aggaatacag cctggagaaa aatgagaggg cctgcattga tttcgccatc 4620 agtgccaagc cgctgacccg acacatgccg cagaacaagc agagcttcca gtaccgcatg 4680 tggcagttcg tggtgtctcc gcctttcgag tacacgatca tggccatgat cgccctcaac 4740 accatcgtgc ttatgatgaa gttctatggg gcttctgtgg cttatgaaaa tgccctgcgg 4800 gtgttcaaca tcgccttcac ctccctcttc tctctggaat gtgtgctgaa agccatggct 4860 tttgggattc tgaattattt ccgcgatgcc tggaacatct tcgactttgt gactgttctg 4920 ggcagcatca ccgatatcct cgtgactgag tttgggaata acttcatcaa cctgagcttt 4980 ctccgcctct tccgagctgc ccggctcatc aaacttctcc gtcagggtta caccatccgc 5040 attcttctct ggacctttgt gcagtccttc aaggccctgc cttatgtctg tctgctgatc 5100 gccatgctct tcttcatcta tgccatcatt gggatgcagg tgtttggtaa cattggcatc 5160 gacgtggagg acgaggacag tgatgaagat gagttccaaa tcactgagca caataacttc 5220 cggaccttct tccaggccct catgcttctc ttccggagtg ccaccgggga agcttggcac 5280 aacatcatgc tttcctgcct cagcgggaaa ccgtgtgata agaactctgg catcctgact 5340 cgagagtgtg gcaatgaatt tgcttatttt tactttgttt ccttcatctt cctctgctcg 5400 tttctgatgc tgaatctctt tgtcgccgtc atcatggaca actttgagta cctcacccga 5460 gactcctcca tcctgggccc ccaccacctg gatgagtacg tgcgtgtctg ggccgagtat 5520 gaccccgcag cttgcggtcg gattcattat aaggatatgt acagtttatt acgagtaata 5580 tctccccctc tcggcttagg caagaaatgt cctcataggg ttgcttgcaa gcggcttctg 5640 cggatggacc tgcccgtcgc agatgacaac accgtccact tcaattccac cctcatggct 5700 ctgatccgca cagccctgga catcaagatt gccaagggag gagccgacaa acagcagatg 5760 gacgctgagc tgcggaagga gatgatggcg atttggccca atctgtccca gaagacgcta 5820 gacctgctgg tcacacctca caagtccacg gacctcaccg tggggaagat ctacgcagcc 5880 atgatgatca tggagtacta ccggcagagc aaggccaaga agctgcaggc catgcgcgag 5940 gagcaggacc ggacacccct catgttccag cgcatggagc ccccgtcccc aacgcaggaa 6000 gggggacctg gccagaacgc cctcccctcc acccagctgg acccaggagg agccctgatg 6060 gctcacgaaa gcggcctcaa ggagagcccg tcctgggtga cccagcgtgc ccaggagatg 6120 ttccagaaga cgggcacatg gagtccggaa caaggccccc ctaccgacat gcccaacagc 6180 cagcctaact ctcagtccgt ggagatgcga gagatgggca gagatggcta ctccgacagc 6240 gagcactacc tccccatgga aggccagggc cgggctgcct ccatgccccg cctccctgca 6300 gagaaccaga ggagaagggg ccggccacgt gggaataacc tcagtaccat ctcagacacc 6360 agccccatga agcgttcagc ctccgtgctg ggccccaagg cccgacgcct ggacgattac 6420 tcgctggagc gggtcccgcc cgaggagaac cagcggcacc accagcggcg ccgcgaccgc 6480 agccaccgcg cctctgagcg ctccctgggc cgctacaccg atgtggacac aggcttgggg 6540 acagacctga gcatgaccac ccaatccggg gacctgccgt cgaaggagcg ggaccaggag 6600 cggggccggc ccaaggatcg gaagcatcga cagcaccacc accaccacca ccaccaccac 6660 catcccccgc cccccgacaa ggaccgctat gcccaggaac ggccggacca cggccgggca 6720 cgggctcggg accagcgctg gtcccgctcg cccagcgagg gccgagagca catggcgcac 6780 cggcagtag 6789 44 2262 PRT human 44 Met Ala Arg Phe Gly Asp Glu Met Pro Ala Arg Tyr Gly Gly Gly Gly 1 5 10 15 Ser Gly Ala Ala Ala Gly Val Val Val Gly Ser Gly Gly Gly Arg Gly 20 25 30 Ala Gly Gly Ser Arg Gln Gly Gly Gln Pro Gly Ala Gln Arg Met Tyr 35 40 45 Lys Gln Ser Met Ala Gln Arg Ala Arg Thr Met Ala Leu Tyr Asn Pro 50 55 60 Ile Pro Val Arg Gln Asn Cys Leu Thr Val Asn Arg Ser Leu Phe Leu 65 70 75 80 Phe Ser Glu Asp Asn Val Val Arg Lys Tyr Ala Lys Lys Ile Thr Glu 85 90 95 Trp Pro Pro Phe Glu Tyr Met Ile Leu Ala Thr Ile Ile Ala Asn Cys 100 105 110 Ile Val Leu Ala Leu Glu Gln His Leu Pro Asp Asp Asp Lys Thr Pro 115 120 125 Met Ser Glu Arg Leu Asp Asp Thr Glu Pro Tyr Phe Ile Gly Ile Phe 130 135 140 Cys Phe Glu Ala Gly Ile Lys Ile Ile Ala Leu Gly Phe Ala Gly His 145 150 155 160 Lys Gly Ser Tyr Leu Arg Asn Gly Trp Asn Val Met Asp Phe Val Val 165 170 175 Val Leu Thr Gly Ile Leu Ala Thr Val Gly Thr Glu Phe Asp Leu Arg 180 185 190 Thr Leu Arg Ala Val Arg Val Leu Arg Pro Leu Lys Leu Val Ser Gly 195 200 205 Ile Pro Ser Leu Gln Val Val Leu Lys Ser Ile Met Lys Ala Met Ile 210 215 220 Pro Leu Leu Gln Ile Gly Leu Leu Leu Phe Phe Ala Ile Leu Ile Phe 225 230 235 240 Ala Ile Ile Gly Leu Glu Phe Tyr Met Gly Lys Phe His Thr Thr Cys 245 250 255 Phe Glu Glu Gly Thr Asp Asp Ile Gln Gly Glu Ser Pro Ala Pro Cys 260 265 270 Gly Thr Glu Glu Pro Ala Arg Thr Cys Pro Asn Gly Thr Lys Cys Gln 275 280 285 Pro Tyr Trp Glu Gly Pro Asn Asn Gly Ile Thr Gln Phe Asp Asn Ile 290 295 300 Leu Phe Ala Val Leu Thr Val Phe Gln Cys Ile Thr Met Glu Gly Trp 305 310 315 320 Thr Asp Leu Leu Tyr Asn Ser Asn Asp Ala Ser Gly Asn Thr Trp Asn 325 330 335 Trp Leu Tyr Phe Ile Pro Leu Ile Ile Ile Gly Ser Phe Phe Met Leu 340 345 350 Asn Leu Val Leu Gly Val Leu Ser Gly Glu Phe Ala Lys Glu Phe Glu 355 360 365 Arg Val Glu Asn Arg Arg Ala Phe Leu Lys Leu Arg Arg Gln Gln Gln 370 375 380 Ile Glu Arg Glu Leu Asn Gly Tyr Met Glu Trp Ile Ser Lys Ala Glu 385 390 395 400 Glu Val Ile Leu Ala Glu Asp Glu Thr Asp Gly Glu Gln Arg His Pro 405 410 415 Phe Asp Gly Ala Leu Arg Arg Thr Thr Ile Lys Lys Ser Lys Thr Asp 420 425 430 Leu Leu Asn Pro Glu Glu Ala Glu Asp Gln Leu Ala Asp Ile Ala Ser 435 440 445 Val Gly Ser Pro Phe Ala Arg Ala Ser Ile Lys Ser Ala Lys Leu Glu 450 455 460 Asn Ser Thr Phe Phe His Lys Lys Glu Arg Arg Met Arg Phe Tyr Ile 465 470 475 480 Arg Arg Met Val Lys Thr Gln Ala Phe Tyr Trp Thr Val Leu Ser Leu 485 490 495 Val Ala Leu Asn Thr Leu Cys Val Ala Ile Val His Tyr Asn Gln Pro 500 505 510 Glu Trp Leu Ser Asp Phe Leu Tyr Tyr Ala Glu Phe Ile Phe Leu Gly 515 520 525 Leu Phe Met Ser Glu Met Phe Ile Lys Met Tyr Gly Leu Gly Thr Arg 530 535 540 Pro Tyr Phe His Ser Ser Phe Asn Cys Phe Asp Cys Gly Val Ile Ile 545 550 555 560 Gly Ser Ile Phe Glu Val Ile Trp Ala Val Ile Lys Pro Gly Thr Ser 565 570 575 Phe Gly Ile Ser Val Leu Arg Ala Leu Arg Leu Leu Arg Ile Phe Lys 580 585 590 Val Thr Lys Tyr Trp Ala Ser Leu Arg Asn Leu Val Val Ser Leu Leu 595 600 605 Asn Ser Met Lys Ser Ile Ile Ser Leu Leu Phe Leu Leu Phe Leu Phe 610 615 620 Ile Val Val Phe Ala Leu Leu Gly Met Gln Leu Phe Gly Gly Gln Phe 625 630 635 640 Asn Phe Asp Glu Gly Thr Pro Pro Thr Asn Phe Asp Thr Phe Pro Ala 645 650 655 Ala Ile Met Thr Val Phe Gln Ile Leu Thr Gly Glu Asp Trp Asn Glu 660 665 670 Val Met Tyr Asp Gly Ile Lys Ser Gln Gly Gly Val Gln Gly Gly Met 675 680 685 Val Phe Ser Ile Tyr Phe Ile Val Leu Thr Leu Phe Gly Asn Tyr Thr 690 695 700 Leu Leu Asn Val Phe Leu Ala Ile Ala Val Asp Asn Leu Ala Asn Ala 705 710 715 720 Gln Glu Leu Thr Lys Asp Glu Gln Glu Glu Glu Glu Ala Ala Asn Gln 725 730 735 Lys Leu Ala Leu Gln Lys Ala Lys Glu Val Ala Glu Val Ser Pro Leu 740 745 750 Ser Ala Ala Asn Met Ser Ile Ala Val Lys Glu Gln Gln Lys Asn Gln 755 760 765 Lys Pro Ala Lys Ser Val Trp Glu Gln Arg Thr Ser Glu Met Arg Lys 770 775 780 Gln Asn Leu Leu Ala Ser Arg Glu Ala Leu Tyr Asn Glu Met Asp Pro 785 790 795 800 Asp Glu Arg Trp Lys Ala Ala Tyr Thr Arg His Leu Arg Pro Asp Met 805 810 815 Lys Thr His Leu Asp Arg Pro Leu Val Val Asp Pro Gln Glu Asn Arg 820 825 830 Asn Asn Asn Thr Asn Lys Ser Arg Ala Ala Glu Pro Thr Val Asp Gln 835 840 845 Arg Leu Gly Gln Gln Arg Ala Glu Asp Phe Leu Arg Lys Gln Ala Arg 850 855 860 Tyr His Asp Arg Ala Arg Asp Pro Ser Gly Ser Ala Gly Leu Asp Ala 865 870 875 880 Arg Arg Pro Trp Ala Gly Ser Gln Glu Ala Glu Leu Ser Arg Glu Asp 885 890 895 Pro Tyr Gly Arg Glu Ser Asp His His Ala Arg Glu Gly Ser Leu Glu 900 905 910 Gln Pro Gly Phe Trp Glu Gly Glu Ala Glu Arg Gly Lys Ala Gly Asp 915 920 925 Pro His Arg Arg His Val His Arg Gln Gly Gly Ser Arg Glu Ser Arg 930 935 940 Ser Gly Ser Pro Arg Thr Gly Ala Asp Gly Glu His Arg Arg His Arg 945 950 955 960 Ala His Arg Arg Pro Gly Glu Glu Gly Pro Glu Asp Lys Ala Glu Arg 965 970 975 Arg Ala Arg His Arg Glu Gly Ser Arg Pro Ala Arg Gly Gly Glu Gly 980 985 990 Glu Gly Glu Gly Pro Asp Gly Gly Glu Arg Arg Arg Arg His Arg His 995 1000 1005 Gly Ala Pro Ala Thr Tyr Glu Gly Asp Ala Arg Arg Glu Asp Lys 1010 1015 1020 Glu Arg Arg His Arg Arg Arg Lys Glu Asn Gln Gly Ser Gly Val 1025 1030 1035 Pro Val Ser Gly Pro Asn Leu Ser Thr Thr Arg Pro Ile Gln Gln 1040 1045 1050 Asp Leu Gly Arg Gln Asp Pro Pro Leu Ala Glu Asp Ile Asp Asn 1055 1060 1065 Met Lys Asn Asn Lys Leu Ala Thr Ala Glu Ser Ala Ala Pro His 1070 1075 1080 Gly Ser Leu Gly His Ala Gly Leu Pro Gln Ser Pro Ala Lys Met 1085 1090 1095 Gly Asn Ser Thr Asp Pro Gly Pro Met Leu Ala Ile Pro Ala Met 1100 1105 1110 Ala Thr Asn Pro Gln Asn Ala Ala Ser Arg Arg Thr Pro Asn Asn 1115 1120 1125 Pro Gly Asn Pro Ser Asn Pro Gly Pro Pro Lys Thr Pro Glu Asn 1130 1135 1140 Ser Leu Ile Val Thr Asn Pro Ser Gly Thr Gln Thr Asn Ser Ala 1145 1150 1155 Lys Thr Ala Arg Lys Pro Asp His Thr Thr Val Asp Ile Pro Pro 1160 1165 1170 Ala Cys Pro Pro Pro Leu Asn His Thr Val Val Gln Val Asn Lys 1175 1180 1185 Asn Ala Asn Pro Asp Pro Leu Pro Lys Lys Glu Glu Glu Lys Lys 1190 1195 1200 Glu Glu Glu Glu Glu Asp Asp Arg Gly Glu Asp Gly Pro Lys Pro 1205 1210 1215 Met Pro Pro Tyr Ser Ser Met Phe Ile Leu Ser Thr Thr Asn Pro 1220 1225 1230 Leu Arg Arg Leu Cys His Tyr Ile Leu Asn Leu Arg Tyr Phe Glu 1235 1240 1245 Met Cys Ile Leu Met Val Ile Ala Met Ser Ser Ile Ala Leu Ala 1250 1255 1260 Ala Glu Asp Pro Val Gln Pro Asn Ala Pro Arg Asn Asn Val Leu 1265 1270 1275 Arg Tyr Phe Asp Tyr Val Phe Thr Gly Val Phe Thr Phe Glu Met 1280 1285 1290 Val Ile Lys Met Ile Asp Leu Gly Leu Val Leu His Gln Gly Ala 1295 1300 1305 Tyr Phe Arg Asp Leu Trp Asn Ile Leu Asp Phe Ile Val Val Ser 1310 1315 1320 Gly Ala Leu Val Ala Phe Ala Phe Thr Gly Asn Ser Lys Gly Lys 1325 1330 1335 Asp Ile Asn Thr Ile Lys Ser Leu Arg Val Leu Arg Val Leu Arg 1340 1345 1350 Pro Leu Lys Thr Ile Lys Arg Leu Pro Lys Leu Lys Ala Val Phe 1355 1360 1365 Asp Cys Val Val Asn Ser Leu Lys Asn Val Phe Asn Ile Leu Ile 1370 1375 1380 Val Tyr Met Leu Phe Met Phe Ile Phe Ala Val Val Ala Val Gln 1385 1390 1395 Leu Phe Lys Gly Lys Phe Phe His Cys Thr Asp Glu Ser Lys Glu 1400 1405 1410 Phe Glu Lys Asp Cys Arg Gly Lys Tyr Leu Leu Tyr Glu Lys Asn 1415 1420 1425 Glu Val Lys Ala Arg Asp Arg Glu Trp Lys Lys Tyr Glu Phe His 1430 1435 1440 Tyr Asp Asn Val Leu Trp Ala Leu Leu Thr Leu Phe Thr Val Ser 1445 1450 1455 Thr Ala Glu Gly Trp Pro Gln Val Leu Lys His Ser Val Asp Ala 1460 1465 1470 Thr Phe Glu Asn Gln Gly Pro Ser Pro Gly Tyr Arg Met Glu Met 1475 1480 1485 Ser Ile Phe Tyr Val Val Tyr Phe Val Val Phe Pro Phe Phe Phe 1490 1495 1500 Val Asn Ile Phe Val Ala Leu Ile Ile Ile Thr Phe Gln Glu Gln 1505 1510 1515 Gly Asp Lys Met Met Glu Glu Tyr Ser Leu Glu Lys Asn Glu Arg 1520 1525 1530 Ala Cys Ile Asp Phe Ala Ile Ser Ala Lys Pro Leu Thr Arg His 1535 1540 1545 Met Pro Gln Asn Lys Gln Ser Phe Gln Tyr Arg Met Trp Gln Phe 1550 1555 1560 Val Val Ser Pro Pro Phe Glu Tyr Thr Ile Met Ala Met Ile Ala 1565 1570 1575 Leu Asn Thr Ile Val Leu Met Met Lys Phe Tyr Gly Ala Ser Val 1580 1585 1590 Ala Tyr Glu Asn Ala Leu Arg Val Phe Asn Ile Ala Phe Thr Ser 1595 1600 1605 Leu Phe Ser Leu Glu Cys Val Leu Lys Ala Met Ala Phe Gly Ile 1610 1615 1620 Leu Asn Tyr Phe Arg Asp Ala Trp Asn Ile Phe Asp Phe Val Thr 1625 1630 1635 Val Leu Gly Ser Ile Thr Asp Ile Leu Val Thr Glu Phe Gly Asn 1640 1645 1650 Asn Phe Ile Asn Leu Ser Phe Leu Arg Leu Phe Arg Ala Ala Arg 1655 1660 1665 Leu Ile Lys Leu Leu Arg Gln Gly Tyr Thr Ile Arg Ile Leu Leu 1670 1675 1680 Trp Thr Phe Val Gln Ser Phe Lys Ala Leu Pro Tyr Val Cys Leu 1685 1690 1695 Leu Ile Ala Met Leu Phe Phe Ile Tyr Ala Ile Ile Gly Met Gln 1700 1705 1710 Val Phe Gly Asn Ile Gly Ile Asp Val Glu Asp Glu Asp Ser Asp 1715 1720 1725 Glu Asp Glu Phe Gln Ile Thr Glu His Asn Asn Phe Arg Thr Phe 1730 1735 1740 Phe Gln Ala Leu Met Leu Leu Phe Arg Ser Ala Thr Gly Glu Ala 1745 1750 1755 Trp His Asn Ile Met Leu Ser Cys Leu Ser Gly Lys Pro Cys Asp 1760 1765 1770 Lys Asn Ser Gly Ile Leu Thr Arg Glu Cys Gly Asn Glu Phe Ala 1775 1780 1785 Tyr Phe Tyr Phe Val Ser Phe Ile Phe Leu Cys Ser Phe Leu Met 1790 1795 1800 Leu Asn Leu Phe Val Ala Val Ile Met Asp Asn Phe Glu Tyr Leu 1805 1810 1815 Thr Arg Asp Ser Ser Ile Leu Gly Pro His His Leu Asp Glu Tyr 1820 1825 1830 Val Arg Val Trp Ala Glu Tyr Asp Pro Ala Ala Cys Gly Arg Ile 1835 1840 1845 His Tyr Lys Asp Met Tyr Ser Leu Leu Arg Val Ile Ser Pro Pro 1850 1855 1860 Leu Gly Leu Gly Lys Lys Cys Pro His Arg Val Ala Cys Lys Arg 1865 1870 1875 Leu Leu Arg Met Asp Leu Pro Val Ala Asp Asp Asn Thr Val His 1880 1885 1890 Phe Asn Ser Thr Leu Met Ala Leu Ile Arg Thr Ala Leu Asp Ile 1895 1900 1905 Lys Ile Ala Lys Gly Gly Ala Asp Lys Gln Gln Met Asp Ala Glu 1910 1915 1920 Leu Arg Lys Glu Met Met Ala Ile Trp Pro Asn Leu Ser Gln Lys 1925 1930 1935 Thr Leu Asp Leu Leu Val Thr Pro His Lys Ser Thr Asp Leu Thr 1940 1945 1950 Val Gly Lys Ile Tyr Ala Ala Met Met Ile Met Glu Tyr Tyr Arg 1955 1960 1965 Gln Ser Lys Ala Lys Lys Leu Gln Ala Met Arg Glu Glu Gln Asp 1970 1975 1980 Arg Thr Pro Leu Met Phe Gln Arg Met Glu Pro Pro Ser Pro Thr 1985 1990 1995 Gln Glu Gly Gly Pro Gly Gln Asn Ala Leu Pro Ser Thr Gln Leu 2000 2005 2010 Asp Pro Gly Gly Ala Leu Met Ala His Glu Ser Gly Leu Lys Glu 2015 2020 2025 Ser Pro Ser Trp Val Thr Gln Arg Ala Gln Glu Met Phe Gln Lys 2030 2035 2040 Thr Gly Thr Trp Ser Pro Glu Gln Gly Pro Pro Thr Asp Met Pro 2045 2050 2055 Asn Ser Gln Pro Asn Ser Gln Ser Val Glu Met Arg Glu Met Gly 2060 2065 2070 Arg Asp Gly Tyr Ser Asp Ser Glu His Tyr Leu Pro Met Glu Gly 2075 2080 2085 Gln Gly Arg Ala Ala Ser Met Pro Arg Leu Pro Ala Glu Asn Gln 2090 2095 2100 Arg Arg Arg Gly Arg Pro Arg Gly Asn Asn Leu Ser Thr Ile Ser 2105 2110 2115 Asp Thr Ser Pro Met Lys Arg Ser Ala Ser Val Leu Gly Pro Lys 2120 2125 2130 Ala Arg Arg Leu Asp Asp Tyr Ser Leu Glu Arg Val Pro Pro Glu 2135 2140 2145 Glu Asn Gln Arg His His Gln Arg Arg Arg Asp Arg Ser His Arg 2150 2155 2160 Ala Ser Glu Arg Ser Leu Gly Arg Tyr Thr Asp Val Asp Thr Gly 2165 2170 2175 Leu Gly Thr Asp Leu Ser Met Thr Thr Gln Ser Gly Asp Leu Pro 2180 2185 2190 Ser Lys Glu Arg Asp Gln Glu Arg Gly Arg Pro Lys Asp Arg Lys 2195 2200 2205 His Arg Gln His His His His His His His His His His Pro Pro 2210 2215 2220 Pro Pro Asp Lys Asp Arg Tyr Ala Gln Glu Arg Pro Asp His Gly 2225 2230 2235 Arg Ala Arg Ala Arg Asp Gln Arg Trp Ser Arg Ser Pro Ser Glu 2240 2245 2250 Gly Arg Glu His Met Ala His Arg Gln 2255 2260 45 20 DNA human 45 caacatcatg ctttcctgcc 20 46 20 DNA human 46 atgatgacgg cgacaaagag 20 47 40 DNA human 47 tctccgcagt cgtagctcca cgcaaaggat gtacaagcag 40 48 41 DNA human 48 ggttgtagag tgccatggtc attcccaagc ctccagggta g 41 49 20 DNA HUMAN 49 acctccaac acccttcttt 20 50 19 DNA HUMAN 50 tctgtgccct gctccactc 19 51 20 DNA HUMAN 51 acgctgacct tgccttctct 20 52 20 DNA HUMAN 52 caaccaaaag cctcgtaatc 20 53 20 DNA HUMAN 53 aaaacccacc ctctgttctc 20 54 20 DNA HUMAN 54 ttgtcagggt cggaaactca 20 55 16 DNA HUMAN 55 cttggtggcg gggttt 16 56 20 DNA HUMAN 56 ctgcctaatc ctcccaagag 20 57 20 DNA HUMAN 57 tcccttccct tttgtagatg 20 58 19 DNA HUMAN 58 gtggggctgt gttgtcctt 19 59 20 DNA HUMAN 59 gacagagcca caagagaacc 20 60 20 DNA HUMAN 60 agcaaagagg agtgagtggg 20 61 20 DNA HUMAN 61 atactctggc ttttctatgc 20 62 20 DNA HUMAN 62 gcatgactct ctttgtactc 20 63 17 DNA HUMAN 63 gcagagaatg ggggtgg 17 64 20 DNA HUMAN 64 ctgaggtggg tttagagcag 20 65 22 DNA HUMAN 65 gggtaacgtc tttttctctt gc 22 66 20 DNA HUMAN 66 atgtctcttg ggcgataggt 20 67 20 DNA HUMAN 67 atttcttctg aaggaacagc 20 68 20 DNA HUMAN 68 ggagggatca gggagttggc 20 69 20 DNA HUMAN 69 caagcctaac ctcctctctg 20 70 19 DNA HUMAN 70 tcattccagg caagagctg 19 71 20 DNA HUMAN 71 atttggaggg aggagtttgg 20 72 20 DNA HUMAN 72 tcactttccc aactttctgg 20 73 20 DNA HUMAN 73 cagaaagttg ggaaagtagc 20 74 19 DNA HUMAN 74 ttgaattcct gtgaaggac 19 75 21 DNA HUMAN 75 cttggagatg agatactgag c 21 76 20 DNA HUMAN 76 caggcacttt catctgtgac 20 77 20 DNA HUMAN 77 tccacagctg catctccaag 20 78 18 DNA HUMAN 78 accctccctt gagcccct 18 79 20 DNA HUMAN 79 cagtggttgc ttttcctgac 20 80 20 DNA HUMAN 80 ttgccagaga aacattctcc 20 81 20 DNA HUMAN 81 tgaacaaaga ttccacgtgc 20 82 21 DNA HUMAN 82 ttcaggagcc agggtagcat c 21 83 59 DNA HUMAN 83 tagcaatgct ctaagtcctg cgcaggagaa ccgcaacaag cagcagggag agccgcagc 59 84 58 DNA HUMAN 84 tgtttcctga ggaagtcctc gcgatgacgt cgatgctcta ccgtcattct gcggattc 58 85 41 DNA HUMAN 85 ggttcttttt cattcacttg cgagaatagc cttatcgtca c 41 86 39 DNA HUMAN 86 tttcctggca gtcttagctg cagtgatgtg agagcagag 39 87 20 DNA HUMAN 87 tgggaaattg tggagggagc 20 88 20 DNA HUMAN 88 tgacttccgc caccctggtg 20 89 21 DNA HUMAN 89 taggaagggg tgtgctctgt g 21 90 20 DNA HUMAN 90 agcctgtggt ctgagtggac 20 91 20 DNA HUMAN 91 atccactgct ctcttgcttt 20 92 22 DNA HUMAN 92 gtggttctca cttataatct gc 22 93 21 DNA HUMAN 93 tggcctcatt ggcttccctg c 21 94 20 DNA HUMAN 94 aagaggaaac ccttgcgaag 20 95 20 DNA HUMAN 95 ctacccaacc tgacctctgc 20 96 20 DNA HUMAN 96 acatgataac cctgacagtc 20 97 20 DNA HUMAN 97 ctcatgctct ctgtcaactc 20 98 20 DNA HUMAN 98 tggttccaat gggaatgtgc 20 99 20 DNA HUMAN 99 ctgcttccca agcagtctag 20 100 20 DNA HUMAN 100 tcctggatag atttccagtc 20 101 20 DNA HUMAN 101 agtttttaaa ggacagatgg 20 102 22 DNA HUMAN 102 tttccctgcc ccattccttt gc 22 103 20 DNA HUMAN 103 ctctgccgct ctcaccactg 20 104 19 DNA HUMAN 104 tttatcaggt agaggcagg 19 105 21 DNA HUMAN 105 ttccaagccc atagctgtag c 21 106 21 DNA HUMAN 106 tgaccctgct actcctgctt c 21 107 20 DNA HUMAN 107 actgtgcctc taacatgcac 20 108 18 DNA HUMAN 108 aagtgctggc tcaagcag 18 109 20 DNA HUMAN 109 tctgtgagtg gtgacagctc 20 110 18 DNA HUMAN 110 gtcacctgtc ttctcagc 18 111 20 DNA HUMAN 111 tggaaggact ctggcacgtg 20 112 20 DNA HUMAN 112 ggaggctctg ggaaccttag 20 113 21 DNA HUMAN 113 agaagccact ggaggaatgg c 21 114 22 DNA HUMAN 114 attatcagag caggtcccct tc 22 115 20 DNA HUMAN 115 tccgagtctc tgatttctcc 20 116 19 DNA HUMAN 116 agacggccct cacagtgtc 19 117 20 DNA HUMAN 117 ttcattccct cggtctctgc 20 118 19 DNA HUMAN 118 ctgactgaac ctgtgagac 19 119 19 DNA HUMAN 119 tgtgaaccca ttgcctgca 19 120 19 DNA HUMAN 120 tgggaatgac tgcgcttgc 19 121 18 DNA HUMAN 121 atgcctggga atgactgc 18 122 18 DNA HUMAN 122 tgtcacgcct gtctgtgc 18 123 18 DNA HUMAN 123 tgacacccag gcaggcag 18 124 18 DNA HUMAN 124 tctgacgcct gtctgtgc 18 125 18 DNA HUMAN 125 ttggtgagct caccgtgt 18 126 21 DNA HUMAN 126 ttcccgtggt gacatgcaag c 21 127 20 DNA HUMAN 127 gtccacacac tgctctctgc 20 128 18 DNA HUMAN 128 acactccacc tccctggc 18 129 18 DNA HUMAN 129 gccagggagg tggagtgt 18 130 18 DNA HUMAN 130 ggttccttcc accgcaac 18 131 17 DNA HUMAN 131 caactcccca atggctc 17 132 21 DNA HUMAN 132 cctacccagt gcagagtgag g 21 133 20 DNA HUMAN 133 tctgtgtgca ccatcccatg 20 134 20 DNA HUMAN 134 aaggattggg ctccatggag 20 135 19 DNA HUMAN 135 gttggtgcta gctgctgac 19 136 20 DNA HUMAN 136 ctttcttctt ccttagtgtc 20 137 20 DNA HUMAN 137 gtgtgctgtc tgaccctcac 20 138 20 DNA HUMAN 138 agcctggggt cacttgcagc 20 139 23 DNA HUMAN 139 cctttgtttc aattttcgtg tag 23 140 20 DNA HUMAN 140 tggggcctgg gtacctccga 20 141 19 DNA HUMAN 141 ctttaattgc cctgtcttc 19 142 18 DNA HUMAN 142 ttaattcgac cacttccc 18 143 20 DNA HUMAN 143 agtgagactc gtctctaatg 20 144 20 DNA HUMAN 144 acctacctga attcctgacc 20 145 22 DNA HUMAN 145 aacactagtg acattatttt ca 22 146 20 DNA HUMAN 146 agctaggcct gaaggcttct 20 

What is claimed is:
 1. An isolated nucleic acid comprising SEQ ID NO:43.
 2. The nucleic acid according to claim 1 comprising a mutation at codon 192 resulting in the replacement of arganine by glutamine.
 3. The nucleic acid according to claim 1 comprising a mutation at codon 666 resulting in the replacement of threonine by methionine.
 4. The nucleic acid according to claim 1 comprising a mutation at codon 714 resulting in the replacement of valine by alanine.
 5. The nucleic acid according to claim 1 comprising a mutation at codon 1811 resulting in a replacement of isoleucine by leucine.
 6. The nucleic acid according to claim 1 comprising a G-to-A mutation at codon
 193. 7. The nucleic acid according to claim 1 comprising an A-to-G mutation at codon
 292. 8. The nucleic acid according to claim 1 comprising a G-to-A mutation at codon
 394. 9. The nucleic acid according to claim 1 comprising a G-to-A mutation at codon 454 resulting in a replacement of alanine by threonine.
 10. The nucleic acid according to claim 1 comprising a C-to-A mutation at codon
 698. 11. The nucleic acid according to claim 1 comprising a G-to-A mutation at codon
 918. 12. The nucleic acid according to claim 1 comprising a T-to-C mutation at codon
 1289. 13. The nucleic acid according to claim 1 comprising a T-to-C mutation at codon
 2221. 14. The nucleic acid according to claim 1 comprising a (CAG)n repeat sequence in its 3′UTR region as indicated in table
 2. 15. An isolated nucleic acid comprising a nucleic acid sequence encoding an amino acid sequence depicted in SEQ ID NO:44. 